Method and kit for detecting mandarinfish iridovirus containing internal reference

Abstract

This invention provides a method and kit for detecting iridoviruses of the genus *Siniperca* containing an internal control. The method includes: obtaining a plasmid containing a viral target gene fragment; configuring a detection reaction system comprising a first primer pair and a first fluorescent probe for specifically amplifying the viral target gene, a second primer pair and a second fluorescent probe for specifically amplifying the host internal control gene, a qPCR master mixture, and nuclease-free water; adding the plasmid, water, and nucleic acid extract of the sample to be tested to the positive control well, negative control well, and sample well of a reaction plate, respectively, and adding the reaction system to the three wells; placing the reaction plate in a quantitative real-time PCR instrument for the reaction, and obtaining the detection results after the reaction. This invention enables qualitative and quantitative analysis of the target virus and precise quality control of sample nucleic acid, laying the foundation for early diagnosis and control of iridoviruses of the genus *Siniperca*.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology for aquatic animal viruses, and in particular to a detection method and kit for iridoviruses of the genus *Sinocyclocheilus* containing an internal control. Background Technology

[0002] Mandarin fish and largemouth bass are core freshwater aquaculture species in my country, but their development is severely constrained by iridovirus diseases. MRV and LMBV both belong to the genus *Ranavirus* of the family Iridoviridae, with linear double-stranded DNA genomes. While their major capsid protein (MCP) genes share highly conserved regions, their host specificity differs significantly, making them both major viral threats in aquaculture production. Current conventional qPCR detection methods do not simultaneously cover both MRV and LMBV, and lack internal control genes for quality control. This leads to false negatives due to factors such as nucleic acid extraction failure and PCR reaction inhibition, affecting detection accuracy.

[0003] Internal control genes are used in qPCR to correct for differences in sample treatment. β-actin, as a eukaryotic housekeeping gene, is stably expressed in the tissues of aquatic animals such as mandarin fish and largemouth bass, making it an ideal internal control choice. Duplex qPCR technology, by using probes with different fluorescent labels in the same reaction system, can simultaneously detect the target gene and the internal control gene, reducing reagent consumption and operational errors, while also verifying the effectiveness of the detection process through the internal control signal. Currently reported MRV and LMBV detection methods mostly do not integrate an internal control system, failing to meet the dual needs of grassroots aquaculture for "accurate detection + quality control." Therefore, developing a universal dual qPCR method containing the internal control β-actin is of great significance for improving the reliability of virus detection and guiding disease prevention and control. Summary of the Invention

[0004] The purpose of this invention is to provide a detection method and kit for iridoviruses of the genus *Morchella* containing an internal control, aiming to address the problems of existing detection methods being unable to detect two viruses simultaneously and prone to false negatives, and to achieve rapid, sensitive, and accurate detection of MRV and LMBV.

[0005] In a first aspect, the present invention provides a method for detecting iridoviruses of the genus *Siniperca* containing an internal control, the method comprising: Step S1: Obtain recombinant plasmid PCE-T-RanaV or PCE-T-Actin plasmid containing viral target gene fragments; Step S2: Configure the detection reaction system, which includes a first primer pair and a first fluorescent probe for specifically amplifying the viral target gene, a second primer pair and a second fluorescent probe for specifically amplifying the host internal reference gene, qPCR reaction master mixture, and nuclease-free water; Step S3: Add plasmid, water, and nucleic acid extract of the sample to be tested to the positive control well, negative control well, and sample well of the reaction plate, respectively, and add the reaction system to the three wells; Step S4: Place the reaction plate in a real-time PCR instrument for reaction, and obtain the detection results after the reaction is completed.

[0006] In some embodiments, the first primer pair includes a forward primer with a sequence as shown in SEQ ID No. 1 and a reverse primer with a sequence as shown in SEQ ID No. 2.

[0007] In some embodiments, the first fluorescent probe sequence is as shown in SEQ ID No. 3, and its 5' end is modified with FAM and its 3' end is modified with BHQ1.

[0008] In some embodiments, the second primer pair includes a forward primer with a sequence as shown in SEQ ID No. 4 and a reverse primer with a sequence as shown in SEQ ID No. 5.

[0009] In some embodiments, the second fluorescent probe sequence is as shown in SEQ ID No. 6, and its 5' end is modified with VIC and its 3' end is modified with BHQ1.

[0010] In some embodiments, the qPCR reaction master mix is ​​NovoStart. Probe qPCR SuperMixII, containing starter Taq DNA polymerase, dNTPs, etc., was purchased from Nearshore Proteins.

[0011] In some embodiments, step S1 includes: Design and synthesize a third primer pair for amplifying a conserved fragment of the major capsid protein gene of the genus *Symplocos spp.* iridovirus; Using the nucleic acid from the positive sample as a template, PCR amplification was performed using the third primer pair described above; The amplification product was cloned into a vector to construct the recombinant plasmid PCE-T-RanaV containing the viral target gene fragment.

[0012] In some embodiments, the third primer pair includes a forward primer with a sequence as shown in SEQ ID No. 7 and a reverse primer with a sequence as shown in SEQ ID No. 8.

[0013] In some embodiments, step S4 includes: If the positive control well has a Ct value of <38 for the FAM and VIC channels and a typical amplification curve, the negative control well has no amplification for the FAM and VIC channels, and the test sample well has a Ct value of <38 for the VIC channel and a typical amplification curve, then the test is valid. Provided the detection is valid, the determination is made based on the Ct value of the FAM channel in the sample well to be tested, specifically: A Ct value < 38 indicates a positive result, a Ct value ≤ 38 < 40 indicates a suspected positive result, and no Ct value indicates a negative result.

[0014] Secondly, this invention proposes a detection kit for iridovirus of the genus *Siniperca spp.* containing an internal reference. The kit includes a first primer pair and a first fluorescent probe for specifically amplifying the viral target gene, a second primer pair and a second fluorescent probe for specifically amplifying the host internal reference gene, a qPCR reaction master mix, and nuclease-free water.

[0015] Compared with the prior art, the present invention has the following advantages: This invention successfully solves the problems of existing technologies, such as the inability to simultaneously detect mandarin fish iridovirus and largemouth bass iridovirus, and the susceptibility to false negatives due to nucleic acid quality or operational errors, by designing a dual-fluorescence quantitative PCR detection method containing a β-actin internal reference gene. Specifically, a recombinant plasmid containing a conserved viral gene (such as the MCP gene) and a host internal reference gene (β-actin) is constructed as a standard. Specific primers and dual-labeled probes (such as a FAM-labeled viral probe and a VIC-labeled internal reference probe) are designed to achieve highly sensitive and specific simultaneous detection of the two viruses and real-time quality control of nucleic acid extraction in the same reaction system. The detection limit of this method reaches 96 copies per microliter, the operation takes only about 60 minutes, and a rigorous negative and positive control system ensures reliable results. This provides an efficient and stable molecular detection tool for the early diagnosis, epidemic monitoring, and precise control of iridovirus in mandarin bass farming. Attached Figure Description

[0016] Figure 1 The PCE-T-RanaV plasmid map; Figure 2 The PCE-T-Actin plasmid map; Figure 3 To detect iridovirus in mandarin fish, primer concentrations of 0.4 μM, 0.35 μM, 0.3 μM, 0.25 μM, 0.2 μM, 0.15 μM, 0.1 μM, 0.05 μM, and a negative control (NTC) were set. When the primer concentration was increased from 0.05 μM to 0.15 μM, the fluorescence value increased significantly. After the concentration exceeded 0.15 μM, the increase in fluorescence value slowed down significantly. Therefore, 0.15 μM was selected as the optimal concentration of the primer.

[0017] Figure 4To detect iridovirus in mandarin fish, probe concentrations of 0.3 μM, 0.25 μM, 0.2 μM, 0.15 μM, 0.1 μM, 0.05 μM, and the corresponding detection results for the negative control (NTC) were set. When the probe concentration was increased from 0.05 μM to 0.1 μM, the fluorescence value increased significantly. After the concentration exceeded 0.1 μM, the increase in fluorescence value slowed down significantly. Therefore, 0.1 μM was selected as the optimal concentration of the probe.

[0018] Figure 5 When detecting the internal control, primer concentrations of 0.4 μM, 0.35 μM, 0.3 μM, 0.25 μM, 0.2 μM, 0.15 μM, 0.1 μM, 0.05 μM, and the corresponding detection results for the negative control (NTC) were set. When the primer concentration was increased from 0.05 μM to 0.2 μM, the fluorescence value increased significantly. After the concentration exceeded 0.2 μM, the increase in fluorescence value slowed down significantly. Therefore, 0.2 μM was selected as the optimal concentration of the primer.

[0019] Figure 6 When detecting the internal control, the probe concentrations were set to 0.3 μM, 0.25 μM, 0.2 μM, 0.15 μM, 0.1 μM, 0.05 μM, and the corresponding detection results for the negative control (NTC). When the probe concentration was increased from 0.05 μM to 0.15 μM, the fluorescence value increased significantly. After the concentration exceeded 0.15 μM, the increase in fluorescence value slowed down significantly. Therefore, 0.15 μM was selected as the optimal concentration of the probe.

[0020] Figure 7 The standard curve of RanaV in Example 5 is shown, with the equation Y = -3.523x + 40.269; Figure 8 The standard curve of β-Actin in Example 6 is shown, with the equation Y = -3.622x + 41.81. Figure 9 This is a specific detection diagram for different viruses and negative controls (NTC) in Example 7 of the present invention. Only the positive controls (PCE-T-RanaV and PCE-T-Actin plasmids) showed amplification at the same time, while only the internal control amplified in the ISKNV, RSIV, and VNNV positive samples, indicating that the method has good specificity. Figure 10 To improve the sensitivity of detecting iridovirus in mandarin fish in Example 10 of this invention, the concentration of the recombinant RanaV plasmid was 9.6 × 10⁻⁶. 4 copies / L, 9.6×10 3 copies / L, 9.6×10 2 copies / L, 9.6×10 1 copies / L, 9.6×10 0 copies / The sensitivity test results for L and the negative control (NTC) show that the lowest detection limit is 9.6 × 10⁻⁶. 1 copies / L; Figure 11 For the detection of internal control sensitivity in Example 9 of this invention, the concentration of the recombinant PCE-T-Actin plasmid is 7.5 × 10⁻⁶. 4 copies / L, 7.5×10 3 copies / L, 7.5×10 2 copies / L, 7.5×10 1 copies / L, 7.5×10 0 copies / The sensitivity test results for L and the negative control (NTC) show that the lowest detection limit is 7.5 × 10⁻⁶. 1 copies / L; Figure 12 The results of detecting MRV positive and negative samples in Example 11 of this invention are as follows: 28 positive samples were tested as positive, and 28 negative samples were tested as negative. Figure 13 The results of detecting LMBV positive and negative samples in Example 11 of this invention are as follows: 28 positive samples were tested as positive and 28 negative samples were tested as negative. Figure 14 The results of the commercial kit used in Example 11 of this invention for detecting MRV positive and negative samples are as follows: 28 positive samples were tested as positive and 28 negative samples were tested as negative. Figure 15 The results of testing LMBV positive and negative samples using the commercially available kit in Example 11 of this invention are as follows: 28 positive samples were tested as positive, and 28 negative samples were tested as negative.

[0021] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art. The terms "comprising" and similar expressions used herein mean that the element or object preceding the word covers the element or object listed after the word and its equivalents, but does not exclude other elements or objects.

[0023] NovoStart in this invention Probe qPCR SuperMix II (UDG) was purchased from Nearshore Proteins Co., Ltd.; amplification primers and probes were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.; this invention uses VirusDNA / RNA Extraction Kit 2.0 purchased from Novizan to obtain pretreated nucleic acids. PCE-T-Actin plasmid and positive samples were preserved in the laboratory.

[0024] 1.1 Construction of positive plasmids PCR primers (third primer pair) were designed targeting conserved sequences of iridoviruses from the genus *Rana spp.* The third primer pair includes RanaV-F and RanaV-R (SEQ NO. 7-SEQ NO. 8), with specific sequences shown in Table 1. After design, the primers were synthesized at Suzhou Genewiz Biotechnology Co., Ltd.

[0025] Table 1. PCR primers for iridoviruses of the genus *Siniperca spp.*

[0026] The positive sample was thawed on ice, and then viral nucleic acid was extracted in a biosafety cabinet according to the operating manual of the Virus DNA / RNA Extraction Kit 2.0.

[0027] The primers for the iridovirus of the genus *Rana spp.* designed in this experiment were used to perform PCR amplification on the nucleic acid sample. The sample was then ligated, transformed, and the recombinant plasmid DH5α strain was screened. The recombinant plasmid was extracted and sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The sequencing results were consistent with the target fragment sequence, indicating that the recombinant plasmid was constructed correctly and named PCE-T-RanaV.

[0028] 1.2 Design and preparation of primers and probes The first primer pair, the first fluorescent probe sequence, the second primer pair, and the second fluorescent probe sequence were designed using Oligo7, as shown in Table 2, and synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0029] Table 2. Primers and probes

[0030] 1.3 Quantitative Fluorescence Detection The detection reaction system is shown in Table 3. After the components in the reaction system are thoroughly mixed, they are amplified in a qPCR instrument according to the program in Table 4.

[0031] Table 3. Detection reaction system

[0032] Table 4. Reaction Procedure

[0033] Example 1: Screening of optimal primers for iridoviruses of the genus *Sinocyclocheilus* In the initial reaction system shown in Table 3, this embodiment only adjusts the volumes of RNase-free ddH2O and the first primer pair, corresponding to the initial reaction system in Table 5. The concentrations of the first primer pair are set to 0.4 μM, 0.35 μM, 0.3 μM, 0.25 μM, 0.2 μM, 0.15 μM, 0.1 μM, and 0.05 μM. That is, the concentration of the first primer pair is changed by adjusting its volume. The sum of the volumes of the first primer pair and the RNase-free ddH2O is 6.8 μL. Furthermore, the initial concentration of the added first primer pair is 10 μL. M controls a single variable; For example, if the concentration of the first primer pair is 0.4 μM, the reaction system includes: 5.2 μL RNase-free ddH2O, 10 μL NovoStart Probe qPCR SuperMix II (UDG), 0.8 μL RanaV-qF, 0.8 μL RanaV-qR, 0.2 μL RanaV-qP, 0.4 μL β-Actin-F, 0.4 μL β-Actin-R, 0.2 μL β-Actin-P, 2.0 μL template; Specifically, the volumes of any primer in the first primer pair are 0.8 μL, 0.7 μL, 0.6 μL, 0.5 μL, 0.4 μL, 0.3 μL, 0.2 μL, and 0.1 μL, respectively, corresponding to volumes of RNase-free ddH2O of 5.2 μL, 5.4 μL, 5.6 μL, 5.8 μL, 6.0 μL, 6.2 μL, 6.4 μL, and 6.6 μL, respectively.

[0034] 2. Finally, add plasmid PCE-T-RanaV and water to the positive control well and negative control well of the reaction plate, respectively, and add the reaction system to the three wells. After capping the tubes, vortex for 30 seconds and centrifuge for 30 seconds.

[0035] 3. Place the reaction plate in the real-time PCR instrument and perform the reaction according to the procedure in Table 4. After the reaction is completed, obtain the detection results.

[0036] Table 5. Initial reaction system

[0037] The results are as follows Figure 3 As shown, the amplification efficiency is high when the primer concentration is 0.15 μM.

[0038] Based on the results obtained in Example 1, a high fluorescence value was observed for the iridovirus gene of the genus *Sinocyclocheilus* when the primer concentration was 0.15 μM. Therefore, primers with a concentration of 0.15 μM were used for subsequent detection.

[0039] Example 2: Screening for optimal iridovirus probes from the genus *Siniperca spp.* In the initial reaction system shown in Table 3, this embodiment only adjusts the volumes of RNase-free ddH2O and the first probe, corresponding to the initial reaction system in Table 5. The concentrations of the first fluorescent probe are set to 0.3 μM, 0.25 μM, 0.2 μM, 0.15 μM, 0.1 μM, and 0.05 μM. That is, the concentration of the first probe is changed by adjusting its volume. The sum of the volumes of the first probe and RNase-free ddH2O is 6.4 μL. Furthermore, the initial concentration of the first probe added is always 10 μL. M controls a single variable; For example, if the concentration of the first probe is 0.3 μM, the reaction system includes: 5.8 μL RNase-free ddH2O, 10 μL NovoStart Probe qPCR SuperMix II (UDG), 0.3μL RanaV-qF, 0.3μL RanaV-qR, 0.6 μLRanaV-qP, 0.4 μL β-Actin-F, 0.4 μL β-Actin-R, 0.2 μL β-Actin-P, 2.0 μL template; Specifically, the volumes of the first probe were 0.6 μL, 0.5 μL, 0.4 μL, 0.3 μL, 0.2 μL, and 0.1 μL, respectively, and the corresponding volumes of RNase-free ddH2O were 5.8 μL, 5.9 μL, 6.0 μL, 6.1 μL, 6.2 μL, and 6.3 μL, respectively.

[0040] 2. Finally, add plasmid PCE-T-RanaV and water to the positive control well and negative control well of the reaction plate, respectively, and add the reaction system to the three wells. After capping the tubes, vortex for 30 seconds and centrifuge for 30 seconds.

[0041] 3. Place the reaction plate in the real-time PCR instrument and perform the reaction according to the procedure in Table 4. After the reaction is completed, obtain the detection results.

[0042] Table 6. Initial reaction system

[0043] The results are as follows Figure 4 As shown, the amplification efficiency is high when the probe concentration is 0.1 μM.

[0044] Based on the results obtained in Example 2, when the probe concentration was 0.1 μM, it showed a high fluorescence value for the detected iridovirus gene of the Mandarin Duck and Bass Frog. Therefore, a probe concentration of 0.1 μM was used for subsequent detection.

[0045] Example 3: Screening of Optimal Internal Reference Primers In the initial reaction system shown in Table 3, this embodiment only adjusted the volume of RNase-free ddH2O and the second primer pair, corresponding to the initial reaction system in Table 5. The concentration of the second primer pair was set to 0.4 μM, 0.35 μM, 0.3 μM, 0.25 μM, 0.2 μM, 0.15 μM, 0.1 μM, and 0.05 μM. That is, the concentration of the second primer pair was changed by adjusting its volume. The sum of the volumes of the second primer pair and RNase-free ddH2O was 7.0 μL. Furthermore, the initial concentration of the added second primer pair was 10 μL. M controls a single variable; For example, if the concentration of the second primer pair is 0.4 μM, the reaction system includes: 5.4 μL RNase-free ddH2O, 10 μL NovoStart Probe qPCR SuperMix II (UDG), 0.3μL RanaV-qF, 0.3μL RanaV-qR, 0.2μL RanaV-qP, 0.8 μL β-Actin-F, 0.8 μL β-Actin-R, 0.2 μL β-Actin-P, 2.0 μL template; Specifically, the volumes of any primer in the second primer pair are 0.8 μL, 0.7 μL, 0.6 μL, 0.5 μL, 0.4 μL, 0.3 μL, 0.2 μL, and 0.1 μL, respectively, corresponding to volumes of RNase-free ddH2O of 5.4 μL, 5.6 μL, 5.8 μL, 6.0 μL, 6.2 μL, 6.4 μL, 6.6 μL, and 6.8 μL.

[0046] 2. Finally, add plasmid PCE-T-Actin and water to the positive control well and negative control well of the reaction plate, respectively, and add the reaction system to the three wells. After capping the tubes, vortex for 30 seconds and centrifuge for 30 seconds.

[0047] 3. Place the reaction plate in the real-time PCR instrument and perform the reaction according to the procedure in Table 4. After the reaction is completed, obtain the detection results.

[0048] Table 7. Initial reaction system

[0049] The results are as follows Figure 5 As shown, the amplification efficiency is high when the primer concentration is 0.2 μM.

[0050] Based on the results obtained in Example 3, when the primer concentration was 0.2 μM, the detection of the internal reference gene showed a high fluorescence value. Therefore, primers with a concentration of 0.2 μM were used for subsequent detection.

[0051] Example 4: Optimal Internal Reference Probe Screening In the initial reaction system shown in Table 3, this embodiment only adjusted the volumes of RNase-free ddH2O and the second probe, corresponding to the initial reaction system in Table 5. The concentrations of the second fluorescent probe were set to 0.3 μM, 0.25 μM, 0.2 μM, 0.15 μM, 0.1 μM, and 0.05 μM. The concentration of the second probe was changed by adjusting its volume. The sum of the volumes of the second probe and RNase-free ddH2O was 6.4 μL. Furthermore, the initial concentration of the added second probe was 10 μL. M controls a single variable; For example, if the concentration of the second probe is 0.3 μM, the reaction system includes: 5.8 μL RNase-free ddH2O, 10 μL NovoStart Probe qPCR SuperMix II (UDG), 0.3μL RanaV-qF, 0.3μL RanaV-qR, 0.2 μLRanaV-qP, 0.4 μL β-Actin-F, 0.4 μL β-Actin-R, 0.6 μL β-Actin-P, 2.0 μL template; Specifically, the volumes of the second probe were 0.6 μL, 0.5 μL, 0.4 μL, 0.3 μL, 0.2 μL, and 0.1 μL, respectively, corresponding to volumes of RNase-free ddH2O of 5.8 μL, 5.9 μL, 6.0 μL, 6.1 μL, 6.2 μL, and 6.3 μL, respectively.

[0052] 2. Finally, add plasmid PCE-T-Actin and water to the positive control well and negative control well of the reaction plate, respectively, and add the reaction system to the three wells. After capping the tubes, vortex for 30 seconds and centrifuge for 30 seconds.

[0053] 3. Place the reaction plate in the real-time PCR instrument and perform the reaction according to the procedure in Table 4. After the reaction is completed, obtain the detection results.

[0054] Table 6. Initial reaction system

[0055] The results are as follows Figure 6 As shown, the amplification efficiency is high when the probe concentration is 0.15 μM.

[0056] Based on the results obtained in Example 4, the probe concentration of 0.15 μM showed a high fluorescence value for the internal reference gene being detected. Therefore, a probe concentration of 0.15 μM was used for subsequent detection.

[0057] Example 5: Standard curve of iridovirus gene from the genus *Siniperca spp.* The strain PCE-T-RanaV-DH5α was revived, and after identification, the recombinant plasmid was extracted and the concentration of the recombinant plasmid was measured to be 243 ng / L. L, converted from molecular weight to copy number, yields a copy number of 9.6 × 10⁻⁶. 10 copies / L, serially diluted 10-fold, yielded 9.6 × 10⁻⁶. 8 copies / L, 9.6×107 copies / L, 9.6×10 6 copies / L, 9.6×10 5 copies / L, 9.6×10 4 copies / L, 9.6×10 3 copies / L, 9.6×10 2 copies / L, 9.6×10 1 copies / A total of 8 samples were collected. qPCR amplification was performed using PCE-T-RanaV recombinant plasmids at the aforementioned 8 concentration gradients as templates. A standard curve was plotted with the logarithm (Log) value of the plasmid concentration on the x-axis and the corresponding Ct value on the y-axis, as shown below. Figure 7 As shown, the Ct value exhibits a good linear relationship with the plasmid concentration, with an amplification efficiency (E) of 92.239% and a correlation coefficient (R) of [missing value]. 2 The value is 0.993.

[0058] Example 6: Standard curve of internal reference gene The recombinant plasmid PCE-T-Actin-DH5α strain was revived, identified, and the recombinant plasmid was extracted. The concentration of the recombinant plasmid was measured to be 165 ng / L. L, converted from molecular weight to copy number, yields a copy number of 7.5 × 10⁻⁶. 10 copies / L, serially diluted 10-fold, yielded 7.5 × 10⁻⁶. 8 copies / L, 7.5×10 7 copies / L, 7.5×10 6 copies / L, 7.5×10 5 copies / L, 7.5×10 4 copies / L, 7.5×10 3 copies / L, 7.5×10 2 copies / L, 7.5×10 1 copies / A total of 8 samples were collected. Using the 8 concentration gradients of PCE-T-Actin recombinant plasmids mentioned above as templates, amplification was performed separately. The reaction systems are shown in Table 3. After thorough mixing, the samples were quantitatively detected using qPCR according to the program in Table 4. A standard curve was plotted with the logarithm (Log) of plasmid concentration as the x-axis and the corresponding Ct value as the y-axis, as shown below. Figure 8 As shown, the Ct value exhibits a good linear relationship with the plasmid concentration, with an amplification efficiency (E) of 88.825% and a correlation coefficient (R). 2 The value is 0.992.

[0059] Example 7: Specificity Test To verify the specificity of the qPCR method, the reaction system was prepared according to Table 3. Infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), and viral neuronecrosis virus (VNNV) nucleic acids were used as templates for amplification. A negative control (ddH2O template) and a positive control (PCE-T-RanaV and PCE-T-Actin plasmid templates) were also set up. After adding the templates, the tubes were capped, vortexed for 30 seconds, centrifuged for 30 seconds, and then placed in a real-time PCR instrument to react according to the procedure in Table 4. The detection results were obtained after the reaction was completed.

[0060] The results obtained in Example 7 are attached. Figure 9 As shown, only the positive controls (PCE-T-RanaV and PCE-T-Actin plasmids) showed simultaneous amplification, indicating that the method has good specificity.

[0061] Example 8: Sensitivity test for iridovirus of the genus *Siniperca spp.* To verify the detection limit of the iridovirus gene in the genus *Sinocyclocheilus*, the strain *PCE-T-RanaV-DH5α* was revived, and after identification, the recombinant plasmid was extracted and the concentration was measured to be 243 ng / L. L, converted from molecular weight to copy number, yielded a copy number of 9.6 × 10⁻¹⁰ after a 10-fold serial dilution. 10 copies / L, serial dilution yielded 9.6 × 10⁻⁶. 4 copies / L, 9.6×10 3 copies / L, 9.6×10 2 copies / L, 9.6×10 1 copies / L, 9.6×10 0 copies / A total of 5 samples were collected. Using the PCE-T-RanaV recombinant plasmids at the above 5 concentration gradients as templates, amplification was performed separately. The reaction systems are shown in Table 3. After thorough mixing, the samples were quantitatively detected using a qPCR instrument according to the program in Table 4.

[0062] The results obtained in Example 8 are as follows Figure 10 As shown, when detecting iridoviruses of the mandarin fish frog genus, a resolution of 9.6 × 10⁻⁶ can be achieved. 1 copies / High-sensitivity detection of L. Among them, 10 4 ~10 1 copies / L-positive plasmids with Ct values ​​in the FAM channel ranging from 24 to 37 were all considered positive; 10 copies / L-positive plasmids (close to the method detection limit) showing a Ct value > 38 or no Ct value in the FAM channel are considered suspected positive. Repeated testing reliably distinguishes low-copy target amplification from background signals. Based on these validation results, assuming effective detection, the Ct value in the FAM channel of the sample well is used as the criterion: Ct value < 38 is considered positive, 38 ≤ Ct value < 40 is considered suspected positive, and no Ct value is considered negative.

[0063] Example 9: Sensitivity test of internal control gene To verify the sensitivity of the internal control gene, the strain PCE-T-Actin-DH5α was revived, identified, and the recombinant plasmid was extracted. The concentration of the recombinant plasmid was measured to be 165 ng / L. L, converted from molecular weight to copy number, yields a copy number of 7.5 × 10⁻⁶. 10 copies / L, serially diluted 10-fold, yielded 7.5 × 10⁻⁶. 4 copies / L, 7.5×10 3 copies / L, 7.5×10 2 copies / L, 7.5×10 1 copies / L, 7.5×10 0 copies / A total of 5 samples were collected. Using the PCE-T-Actin recombinant plasmids at the above 5 concentration gradients as templates, amplification was performed separately. The reaction systems are shown in Table 3. After thorough mixing, the samples were quantitatively detected using a qPCR instrument according to the program in Table 4.

[0064] The results obtained in Example 9 are as follows Figure 11 As shown, when detecting the internal parameters, a value of 7.5 × 10⁻⁶ can be achieved. 1 copies / High-sensitivity detection of L.

[0065] Example 10: Repeatability Test To verify the repeatability of the method, intra-batch and inter-batch repeatability tests were conducted. The procedure is as follows: Standard recombinant plasmids of different concentrations were selected, and four concentration gradients (9.67 × 10⁻⁶) were set for the PCE-T-RanaV plasmid. 6 copies / L, 9.67×10 5 copies / L, 9.67×10 4 copies / L, 9.67×10 3 copies / L), PCE-T-Actin plasmid was configured with 4 concentration gradients (7.55 × 10). 6 copies / L, 7.55×10 5 copies / L, 7.55×10 4 copies / L, 7.55×10 3 copies / L). Intra-batch repeatability: Under the same conditions and in the same batch, three parallel samples were set up for each concentration, and the tests were performed according to the reaction system in Table 3 and the procedure in Table 4. Inter-batch repeatability: The above tests were repeated using three batch reaction systems. The Ct values ​​of each group were recorded, and the coefficient of variation (CV) was calculated to evaluate the intra-batch and inter-batch stability of this qPCR method.

[0066] The PCE-T-RanaV plasmid detection results showed that the intra-batch coefficient of variation was 0.69%–3.39%, and the inter-batch coefficient of variation was 1.45%–4.47% (Table 5). The PCE-T-Actin plasmid detection results showed that the intra-batch coefficient of variation was 0.28%–1.27%, and the inter-batch coefficient of variation was 0.08%–2.44% (Table 6), both of which were less than 5%, indicating that the method has good reproducibility.

[0067] Table 5. Repeatability test of mandarin fish iridovirus

[0068] Table 6. Repeatability Tests of Internal Controls

[0069] Example 11: Clinical Sample Validation The method established according to this invention was used to detect 28 clinically positive MRV samples, 28 clinically negative MRV samples, 28 clinically positive LMBV samples, and 28 clinically negative LMBV samples. The results were compared with two commercially available MRV and LMBV kits. The reaction system is shown in Table 3. After thorough mixing, the samples were subjected to quantitative fluorescence detection in a qPCR instrument according to the program in Table 4.

[0070] The results obtained in Example 11 are shown in Tables 7 and 8. Figure 12 , 13 As shown in Figures 14 and 15, the positive and negative concordance rates for MRV and LMBV were both 100%.

[0071] Table 7. Comparison results with commercial MRV kits

[0072] Table 8. Comparison results with LMBV commercial kits

[0073] Results Analysis: According to Examples 1 to 4 provided by the present invention, through systematic optimization of primer and probe concentrations, the optimal primer concentration for detecting *Rhizophora mandarinata* iridovirus was determined to be 0.15 μM and the optimal probe concentration to be 0.1 μM, while the optimal primer concentration for the internal reference gene was determined to be 0.2 μM and the optimal probe concentration to be 0.15 μM, significantly improving amplification efficiency and fluorescence signal intensity. Based on this, the standard curves constructed according to Examples 5 and 6 showed that the cycle threshold and plasmid copy number of the viral target gene and the internal reference gene exhibited a good linear relationship, with correlation coefficients reaching 0.993 and 0.992, respectively, and amplification efficiencies of 92.24% and 88.83%, respectively, indicating accurate and reliable quantification. The specificity test results of Example 7 showed that only the positive control showed specific amplification, while other common aquatic animal viruses such as infectious spleen and kidney necrosis virus, red sea bream iridovirus, and viral neuronecrosis virus showed no non-target amplification, confirming the high specificity of this method. Based on the sensitivity tests in Examples 8 and 9, the detection limits for the viral target gene and the internal reference gene are shown to be 9.6 × 10⁻⁶, respectively. 1 copies / L and 7.5×10 1 copies / L demonstrates excellent detection sensitivity. In the repeatability test of Example 10, both intra- and inter-batch coefficients of variation were less than 5%, proving the good stability of the method. According to the clinical sample validation results of Example 11, the method of the present invention and the commercial kit showed a 100% concordance rate in positive and negative samples of mandarin fish iridovirus and largemouth bass iridovirus, fully validating its accuracy and reliability in real sample detection.

[0074] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for detecting iridoviruses in the genus *Siniperca spp.* containing an internal control, characterized in that... The method includes: Step S1: Obtain recombinant plasmid PCE-T-RanaV or PCE-T-Actin plasmid containing viral target gene fragments; Step S2: Configure the detection reaction system, which includes a first primer pair and a first fluorescent probe for specifically amplifying the viral target gene, a second primer pair and a second fluorescent probe for specifically amplifying the host internal reference gene, qPCR reaction master mixture, and nuclease-free water; Step S3: Add plasmid, water, and nucleic acid extract of the sample to be tested to the positive control well, negative control well, and sample well of the reaction plate, respectively, and add the reaction system to the three wells; Step S4: Place the reaction plate in a real-time PCR instrument for reaction, and obtain the detection results after the reaction is completed.

2. The detection method for iridovirus of the genus *Siniperca* containing an internal reference according to claim 1, characterized in that, The first primer pair includes a forward primer with a sequence as shown in SEQ ID No. 1 and a reverse primer with a sequence as shown in SEQ ID No.

2.

3. The detection method for iridovirus of the genus *Siniperca* containing an internal reference according to claim 1, characterized in that, The first fluorescent probe sequence is shown in SEQ ID No. 3, and its 5' end is modified with FAM and its 3' end is modified with BHQ1.

4. The detection method for iridovirus of the genus *Siniperca* containing an internal reference according to claim 1, characterized in that, The second primer pair includes a forward primer with a sequence as shown in SEQ ID No. 4 and a reverse primer with a sequence as shown in SEQ ID No.

5.

5. The detection method for iridovirus of the genus *Siniperca* containing an internal reference according to claim 1, characterized in that, The second fluorescent probe sequence is shown in SEQ ID No. 6, and its 5' end is modified with VIC and its 3' end is modified with BHQ1.

6. The detection method for iridovirus of the genus *Siniperca* containing an internal reference according to claim 1, characterized in that, The master mix for the qPCR reaction was NovoStart. Probe qPCR SuperMix II.

7. The detection method for iridovirus of the genus *Siniperca* containing an internal reference according to claim 1, characterized in that, Step S1 includes: Design and synthesize a third primer pair for amplifying a conserved fragment of the major capsid protein gene of the genus *Symplocos spp.* iridovirus; Using the nucleic acid from the positive sample as a template, PCR amplification was performed using the third primer pair described above; The amplification product was cloned into a vector to construct the recombinant plasmid PCE-T-RanaV containing the viral target gene fragment.

8. The method for detecting iridoviruses of the genus *Siniperca* containing an internal reference according to claim 7, characterized in that, The third primer pair includes a forward primer with a sequence as shown in SEQ ID No. 7 and a reverse primer with a sequence as shown in SEQ ID No.

8.

9. The method for detecting iridoviruses of the genus *Siniperca* containing an internal reference according to claim 1, characterized in that, Step S4 includes: If the positive control well has a Ct value of <38 for the FAM and VIC channels and a typical amplification curve, the negative control well has no amplification for the FAM and VIC channels, and the test sample well has a Ct value of <38 for the VIC channel and a typical amplification curve, then the test is valid. Provided the detection is valid, the determination is made based on the Ct value of the FAM channel in the sample well to be tested, specifically: A Ct value < 38 indicates a positive result, a Ct value ≤ 38 < 40 indicates a suspected positive result, and no Ct value indicates a negative result.

10. A detection kit for iridoviruses of the genus *Siniperca* containing an internal control, characterized in that, The kit includes a first primer pair and a first fluorescent probe for specifically amplifying viral target genes, a second primer pair and a second fluorescent probe for specifically amplifying host internal reference genes, a qPCR reaction master mix, and nuclease-free water.

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