Primer combination for detecting larimichthys crocea iridovirus, kit and cdPCR detection method

Abstract

The invention belongs to the technical field of aquatic pathogen detection, and provides a primer and probe combination with strong specificity and high sensitivity, a kit and a cdPCR detection method in order to solve the problems of dependence of absolute quantification of an existing qPCR technology on a standard curve and low repeatability and stability caused by uncontrollable quality of a standard substance. The primer probe can generate specific amplification on LYCIV and has no cross reaction on other common aquatic pathogens (such as NNV, DIV1 and the like), so that the specificity of a detection result is fundamentally ensured, a false positive result caused by the cross reaction is effectively avoided, and the diagnosis accuracy is improved. The method has the characteristics of extremely high sensitivity and absolute quantification, the lower limit of detection reaches 6.2 copies / mu L, the method has more advantages in quantification of samples with extremely low concentration, early diagnosis and detection of extremely low virus load can be realized, viruses with extremely low content in fish bodies can be detected earlier, precious time is provided for disease early warning and early intervention, and the method is worthy of popularization and application. Disease outbreak is effectively prevented.

Description

Technical Field

[0001] This invention belongs to the field of aquatic pathogen detection technology, and more specifically relates to a primer combination, reagent kit, and cdPCR detection method for detecting iridovirus in large yellow croaker. Background Technology

[0002] Large yellow croaker (Larimichthyscrocea) is an important marine aquaculture fish in my country. Large yellow croaker iridovirus disease is a viral disease caused by large yellow croaker iridovirus (LYCIV) of the genus Leptocytovirus in the family Iridoviridae. It was first reported by He Aihua et al., and later confirmed by Chen Xinhua et al. as the pathogen causing large-scale mortality of farmed large yellow croaker juveniles in Ningde and Luoyuan, Fujian Province, during the summer of 1999-2001. This disease has become a major threat to large yellow croaker during the high-temperature summer season, causing huge economic losses to the aquaculture industry. Since there is currently no effective treatment, early and accurate monitoring and diagnosis are crucial for prevention and control.

[0003] Currently, laboratory detection methods for LYCIV mainly rely on polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). qPCR, in particular, has become the primary detection method due to its high sensitivity, strong specificity, and ability to achieve quantitative detection. However, existing qPCR technologies have inherent technical defects and limitations in practical applications. For example, as can be seen from the RAA isothermal fluorescence detection method and reagents for large yellow croaker iridovirus (LYCIV) disclosed in Chinese invention patent application CN110592274A, and the dual TaqMan probe method quantitative real-time PCR detection kit and preparation method for large yellow croaker iridovirus disclosed in Chinese invention patent application CN111979356A, absolute quantification by qPCR requires plotting a standard curve using a series of positive standards of known concentrations to estimate the viral load in the sample. The quantitative results of this method heavily depend on the accurate quantification and stability of the standards. Any errors in the preparation, storage, and dilution of standards, as well as differences in amplification efficiency between different batches of experiments, will directly lead to deviations in the quantitative results, affecting the accuracy and reliability of the detection. The batch-to-batch reproducibility of qPCR is poor. Due to the dependence on standards and amplification efficiency mentioned above, the results of experiments (i.e., batch-to-batch reproducibility) conducted by qPCR at different times, by different operators, and with different reagent batches fluctuate greatly, making it difficult to guarantee the stability and comparability of the test results. This brings difficulties to long-term disease monitoring and epidemiological investigation.

[0004] Therefore, within the technical framework of qPCR, its quantitative principle dictates that it cannot escape its dependence on standard curves. Although errors can be reduced through careful preparation of standards and optimization of reaction conditions, this systemic problem cannot be fundamentally solved. Therefore, there is an urgent need for a detection technology that can achieve absolute quantification without relying on standard curves, and possesses higher repeatability and stability, to address the pressing need for accurate LYCIV monitoring.

[0005] Digital PCR (cdPCR) is a new generation of nucleic acid quantification technology developed in recent years. Its principle involves dividing the reaction system into tens of thousands of microreaction units. After endpoint PCR amplification, the absolute copy number of the target gene is calculated directly based on the Poisson distribution principle by counting the number of positive droplets, thus achieving absolute quantification independent of a standard curve. This technology has demonstrated significant advantages in clinical diagnostics and microbial detection due to its extremely high sensitivity, accuracy, and repeatability. However, to date, no mature method for applying cdPCR technology to the detection of large yellow croaker iridovirus (LYCIV) has been publicly reported. Summary of the Invention

[0006] In view of the above-mentioned technical problems, the present invention provides a primer combination, kit, and cdPCR detection method for detecting large yellow croaker iridovirus (LYCIV). The primary objective of the present invention is to provide a primer and probe combination with high specificity and sensitivity. This primer and probe combination is designed for the conserved region of the LYCIV MCP gene, which can provide a basis for accurate identification and signal amplification for subsequent cdPCR detection, thereby fundamentally ensuring the specificity and reliability of the detection method.

[0007] A second objective of this invention is to provide a kit containing the aforementioned primer combination. This kit integrates the core components required for detection, making it a standardized and commercially available product that is easy to promote and apply in actual testing laboratories, ensuring the standardization of the testing process and the comparability of results.

[0008] The third objective of this invention is to establish a cdPCR-based absolute quantitative detection method for LYCIV. This method overcomes the problems of low accuracy and poor repeatability inherent in existing qPCR techniques, which rely on standard curves for quantification, by microdropletizing the reaction system and directly counting the virus copies using the Poisson distribution principle. Simultaneously, the method maintains low intra- and inter-batch repeatability coefficients of variation, significantly improving the stability and reliability of the detection results and meeting the data comparability requirements for long-term disease monitoring. Furthermore, this method can accurately detect extremely low concentrations of LYC virus in the sample. Through the high sensitivity of cdPCR technology, the detection limit can reach the order of single copies, providing a powerful technical tool for monitoring early infection and the incubation period of the virus.

[0009] The fourth objective of this invention is to clarify the application of primer combinations and reagent kits in the preparation of products for diagnosing iridovirus disease in large yellow croaker. This will provide a clear basis and application direction for the final implementation of this technology, serving disease prevention and control and clinical diagnosis in aquaculture.

[0010] To achieve the above objectives, in a first aspect, the present invention provides a primer set for detecting iridovirus in large yellow croaker, the primer set comprising specific primers and probes, the specific primers comprising an upstream primer with a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 2, and the probe having a nucleotide sequence as shown in SEQ ID No. 3.

[0011] Preferably, the probe is labeled with a fluorescent reporter group at its 5' end and a quencher group at its 3' end. Preferably, the fluorescent reporter group is VIC and the quencher group is BHQ1.

[0012] A second aspect of the present invention provides a kit for detecting iridovirus in large yellow croaker by cdPCR, comprising the primer combination described in the first aspect of the present invention.

[0013] In some preferred embodiments, the kit further includes dPCR premix, consumables for fabricating cdPCR chips, and / or positive control materials;

[0014] Preferably, the positive control is a recombinant plasmid containing the LYCIV MCP gene, the major capsid protein gene of large yellow croaker iridovirus.

[0015] A third aspect of the present invention provides a method for detecting iridovirus in large yellow croaker using cdPCR, comprising the following steps:

[0016] S1. Extract genomic DNA from the sample to be tested;

[0017] S2. Prepare a cdPCR reaction system, wherein the cdPCR reaction system comprises the primer combination, dPCR premix, and genomic DNA of the sample to be tested as described in the first aspect of the present invention;

[0018] S3. Add the cdPCR reaction system to the cdPCR chip, seal the slide, and amplify it using a microarray.

[0019] S4. Use a chip reader to read the amplified fluorescence signal and calculate the absolute copy number of large yellow croaker iridovirus in the test sample based on the number of positive droplets.

[0020] In a further preferred embodiment, the cdPCR reaction system described in step S2, in a total volume of 20 μL, comprises:

[0021] 10×dPCRMix 2.0 μL;

[0022] For example, 1.0 μL of the upstream primer shown in SEQ ID NO.1 has a concentration of 10 μmol / L;

[0023] For example, 1.0 μL of the downstream primer shown in SEQ ID NO.2 has a concentration of 10 μmol / L;

[0024] The probe shown in SEQ ID NO.3 is 1.0 μL, with a concentration of 10 μmol / L;

[0025] The sample to be tested contained 2.0 μL of genomic DNA template.

[0026] The remainder is nuclease-free water.

[0027] In other preferred embodiments, the amplification procedure in step S3 is as follows:

[0028] Incubate at 50°C for 10 minutes; pre-denature at 95°C for 5 minutes; then perform 45 cycles of amplification, each cycle including denaturation at 95°C for 30 seconds and annealing / extension at 56°C for 45 seconds; finally store at 4°C.

[0029] The coefficients of variation for both intra-batch and inter-batch repeatability tests of the method for detecting iridovirus in large yellow croaker using cdPCR in this invention are less than 8.1%.

[0030] More preferably, the detection limit of the method for detecting iridovirus in large yellow croaker using cdPCR in this invention is 6.2 copies / μL.

[0031] Furthermore, a fourth aspect of the present invention provides the use of primer combinations as described in the first aspect of the present invention, or kits as described in the second aspect of the present invention, in the preparation of samples for detecting iridovirus disease in large yellow croaker.

[0032] Unlike existing technologies, the above-mentioned technical solution combines highly specific primers and probes with cdPCR technology, completely eliminating the need for qPCR to use standards to construct standard curves for quantitative estimation. This eliminates systematic errors caused by inaccurate standard preparation, storage, dilution, and fluctuations in amplification efficiency, resulting in more accurate and reliable quantitative results. The primers and probes specifically amplify LYCIV while showing no cross-reactivity with other common aquatic pathogens (such as NNV and DIV1), fundamentally ensuring the specificity of the detection results and effectively avoiding false positives caused by cross-reactivity, thus improving diagnostic accuracy. Furthermore, this invention's technical solution possesses extremely high sensitivity and absolute quantification, with a detection limit of 6.2 copies / μL. It is particularly advantageous for quantifying extremely low concentration samples, enabling early diagnosis and detection of extremely low viral loads. This allows for earlier detection of viruses at very low levels in fish, providing valuable time for disease warning and early intervention, effectively preventing disease outbreaks.

[0033] Furthermore, through optimized reaction systems and conditions, the method for detecting iridovirus in large yellow croaker using cdPCR in this invention exhibits a coefficient of variation (CV) of less than 8.1% in both intra-batch and inter-batch repeatability tests. This ensures the consistency and comparability of test results across different times, operators, and laboratories, which is crucial for long-term epidemiological tracking, vaccine efficacy evaluation, and assessment of control measures. Regardless of whether the sample to be tested is at a high or low concentration, accurate quantitative results can be obtained using the detection method of this invention, resulting in wider applicability and higher quantitative precision.

[0034] Furthermore, the reagent kit provided by this invention integrates optimized core components and, together with standardized operating procedures, simplifies experimental steps, reduces operational difficulty and technical requirements for personnel, which is conducive to the standardized promotion and widespread application of this method, and is particularly suitable for use in grassroots testing units and large-scale screening scenarios.

[0035] The above description of the invention is merely an overview of the technical solution of this application. In order to enable those skilled in the art to better understand the technical solution of this application and to implement it based on the description and drawings, and to make the above-mentioned objectives and other objectives, features and advantages of this application easier to understand, the following description is provided in conjunction with the specific embodiments and drawings of this application. Attached Figure Description

[0036] The accompanying drawings are only used to illustrate the principles, implementation methods, applications, features, and effects of specific embodiments of this application and other related content, and should not be considered as limitations on this application. In the accompanying drawings:

[0037] Figure 1The PCR identification results of the recombinant plasmid standard described in the specific implementation embodiment are shown below, where M is DL2000 DNA Marker; 1 is DEPC water; and 2 is recombinant plasmid LYCIV-MCP.

[0038] Figure 2 The specific test results of cdPCR described in the specific implementation method are shown below, where the X-axis represents the cdPCR detection result; the Y-axis represents the gray value of the HEX channel; gray scatter dots represent negative wells, and green scatter dots represent positive wells.

[0039] Figure 3 The results of the cdPCR sensitivity test described in the specific implementation method are shown below. In this method, the X-axis represents the cdPCR detection result; the Y-axis represents the gray value of the HEX channel; gray dots represent negative wells, and green dots represent positive wells.

[0040] Figure 4 The standard curve for cdPCR described in the specific implementation method is shown, where the X-axis represents the actual copy number of the detected sample (copies / μL), and the Y-axis represents the copy number of the plasmid standard (copies / μL). Detailed Implementation

[0041] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.

[0042] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0043] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0044] In the description of this application, the term "and / or" is used to describe the logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and A and B exist simultaneously. Additionally, the character " / " in this document generally indicates that the preceding and following objects have an "or" logical relationship.

[0045] In this application, terms such as “first” and “second” are used only to distinguish one entity or operation from another, and do not necessarily require or imply any actual quantity, hierarchy or order relationship between these entities or operations.

[0046] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.

[0047] As understood in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceeding" are understood to exclude the stated number; expressions such as "above," "below," and "within" are understood to include the stated number. Furthermore, in the description of the embodiments in this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times," unless otherwise explicitly specified.

[0048] In the description of the embodiments of this application, the space-related expressions used, such as "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "vertical," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential," indicate the orientation or positional relationship based on the orientation or positional relationship shown in the specific embodiments or drawings. They are only for the purpose of describing the specific embodiments of this application or for the reader's understanding, and do not indicate or imply that the device or component referred to must have a specific position, a specific orientation, or be constructed or operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.

[0049] Unless otherwise expressly specified or limited, the terms "installation," "connection," "linking," "fixing," and "setting," as used in the description of the embodiments of this application, should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral setting; it can be a mechanical connection, an electrical connection, or a communication connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be the internal connection of two components or the interaction between two components. For those skilled in the art to which this application pertains, the specific meaning of the above terms in the embodiments of this application can be understood according to the specific circumstances.

[0050] Unless otherwise specified, the experimental reagents used in the embodiments of the present invention are all conventional molecular biology reagents in the art and can be purchased through commercial channels.

[0051] The cdPCR detection method established in this invention can be implemented on a common chip-based digital PCR system in this technical field, such as the digital PCR20K chip system (including a chip, cdPCR 10× premixed solution, sample preparation instrument, PCR amplification instrument, and biochip reader) from Zizhenzhun Biotechnology (Shanghai) Co., Ltd. Those skilled in the art can adaptively adjust non-critical parameters such as sample loading volume and droplet generation conditions according to the operating manual of the selected system.

[0052] The main instruments and equipment used in the embodiments of this invention are as follows: a micro-volume nucleic acid and protein analyzer (DENOVIX, DS-11), a PCR amplification instrument (Zhenzhun Biotechnology, 20D0058SH), and a chip reader (Zhenzhun Biotechnology, RB2110002). Unless otherwise specified, other instruments and equipment can be those conventionally used in the field.

[0053] Example 1: Construction of Recombinant Plasmid Standards

[0054] Materials: LYCIV positive samples were provided by Fujian Provincial Fisheries Technology Extension Station; pESI-T cloning vector was purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0055] Target gene amplification: Using LYCIV genomic DNA as a template, PCR amplification was performed using primers MCP-F (SEQ ID No. 4: 5'-ATGTCTGCAATCTCAGGTGAAAACGTAA-3') and MCP-R (SEQ ID No. 5: 5'-CAGGATAGGGAAGCCTGCGGCG-3'). Reaction program: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 90 s, for a total of 35 cycles; final extension at 72℃ for 5 min. The amplified products were detected by agarose gel electrophoresis, showing a specific band at approximately 1360 bp.

[0056] Cloning and Sequencing: The purified PCR product was cloned into the pESI-T vector, transformed into competent DH5α cells, and positive clones were selected for culture and plasmid extraction. The extracted plasmids were then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the LYCIV MCP gene sequence in GeneBank (accession number: OK149103.1). The recombinant plasmid with a completely identical sequence was named pESI-T-LYCIV-MCP, which served as the required recombinant plasmid standard.

[0057] Copy number calculation: The plasmid concentration was determined using an ultra-micro nucleic acid protein analyzer, and the result was calculated according to the formula: Copy number (copies / μL) = [plasmid concentration (ng / μL) × 10⁻] 9 Calculate the standard copy number concentration of the plasmid using [×6.02×10²³] / [plasmid length (bp)×660], aliquot and store at -80℃ for later use.

[0058] Example 2: Design and Synthesis of Primers and Probes

[0059] Based on the conserved sequence of the LYCIV MCP gene (OK149103.1), specific primers and TaqMan probes were designed using PrimerPremier5.0 software.

[0060] Upstream primer (LYCIV-MCP-F): 5'-GTGAGGAGCGTGAGGTCGTG-3' (SEQ ID NO.1)

[0061] Downstream primer (LYCIV-MCP-R): 5'-TGCCGGTGGGTGACATTCTTG-3' (SEQ ID NO.2)

[0062] Probe (LYCIV-MCP-Probe): 5'-VIC-CAGTCTAGCCGTAGCATGCTCAT-BHQ1-3' (SEQ IDNO.3)

[0063] The probe is labeled with a VIC fluorescent reporter group at its 5' end and a BHQ1 quencher group at its 3' end.

[0064] All primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and purified by HPLC.

[0065] Example 3 Specificity Test

[0066] To verify the specificity of the method of the present invention, nucleic acids of various common aquatic pathogens, such as LYCIV, neuronecrosis virus (NNV), Decapoda iridovirus 1 (DIV1), Pseudomonas plecoglossicida, and Vibrio harveyi, were used as templates for detection using the cdPCR method established in this invention.

[0067] The results are as follows Figure 2 As shown, the channels from left to right represent samples of large yellow croaker iridovirus (LYCIV), neuronecrosis virus (NNV), decapod iridovirus 1 (DIV1), *Pseudomonas plecoglossicida*, and *V. harvestyi*. However, a large number of positive droplets were detected only in the LYCIV template, while the detection results for other pathogens were negative. This indicates that the primer and probe combination used in this invention has high specificity for large yellow croaker iridovirus (LYCIV) and shows no cross-reactivity with other common aquatic pathogens.

[0068] Example 4: Sensitivity and Linear Range Test

[0069] The pESI-T-LYCIV-MCP recombinant plasmid standard prepared in Example 1 was serially diluted 10-fold to obtain a series of standards with concentrations ranging from 10³ copies / μL to 10⁻¹ copies / μL. cdPCR was performed using these standards as templates, with each concentration repeated three times.

[0070] The digital chip, after PCR amplification, is placed in a reader analyzer to read the chip, thus obtaining the LYCIV MCP gene copy number per μL of reaction solution. The nucleic acid copy number in the original sample is calculated using the formula: Sample copy number concentration (copies / μL) = MCP gene copy number per μL of reaction solution (copies / μL) × Total reaction volume (μL) / Template volume (μL). A standard curve is then constructed with the actual copy number of the amplified product as the x-axis and the theoretical copy number of the plasmid standard as the y-axis.

[0071] Please see Figure 3 The results in this figure show that, from left to right, the channels represent concentrations of 10... -1 10 0 10 1 10 2 10 3 LYCIV-MCP plasmid standards at copies / μL. As shown in the figure, the limit of detection (LoD) of the method of this invention is 6.2 copies / μL, exhibiting high sensitivity and good linearity in the range of 10³ copies / μL to 10⁻¹ copies / μL. Figure 4 The linear regression equation was y = 0.6092x + 8.931, with a correlation coefficient R² = 0.9985 (y is the theoretical copy number logarithm, and x is the measured copy number logarithm). These results indicate that the cdPCR method established in this invention exhibits good linearity and high sensitivity for LYCIV MCP plasmid standards.

[0072] Example 5 Repeatability Test

[0073] To evaluate the reproducibility of the method of the present invention, three different concentration gradients of LYCIV genomic DNA were selected as templates.

[0074] Intra-batch repeatability: Within the same batch, each concentration template is tested three times simultaneously.

[0075] Inter-batch repeatability: Select one medium-concentration template and perform one test per day for three consecutive days, with three replicates per test.

[0076] The mean, standard deviation, and coefficient of variation (CV) of each group of data were calculated. The results are shown in Table 1. The CV of intra-batch replicates at high concentration (58779.87±1022.62 copies / μL), medium concentration (747.77±22.63 copies / μL), and low concentration (18.70±1.51 copies / μL) were all less than 8.10%, and the CV of inter-batch replicates within the medium concentration gradient range was 5.62%, indicating that the cdPCR method established in this invention has excellent repeatability and stability.

[0077]

[0078] Example 6: Detection of Clinical Samples

[0079] To verify the practicality and accuracy of the method of this invention, nucleic acid from 21 large yellow croaker tissue samples preserved in the Fujian Provincial Fisheries Technology Extension Station laboratory in 2024 was used as templates, and the cdPCR method established in this invention was employed for detection. Since there is currently no detection standard for LYCIV pathogen, this embodiment also uses the qPCR method reported in Chinese invention patent application CN1827778A to simultaneously detect the above samples, compares the detection results of the two methods, and calculates their concordance rate.

[0080] The cdPCR method established in this invention and the qPCR method reported in Chinese invention patent application CN1827778A were used to detect nucleic acid in 21 large yellow croaker tissue samples preserved in our laboratory in 2024. The results are shown in Table 2. The cdPCR detection results were consistent with the qPCR detection results, with a concordance rate of 100%. In both cases, 10 samples were positive and 11 samples were negative.

[0081] The method for detecting iridovirus in large yellow croaker using cdPCR established in this embodiment specifically includes the following steps:

[0082] S1. Extract genomic DNA from the sample to be tested according to standard procedures;

[0083] S2. Prepare a cdPCR reaction system. The cdPCR reaction system includes a primer combination consisting of an upstream primer with the nucleotide sequence shown in SEQ ID No. 1, a downstream primer with the nucleotide sequence shown in SEQ ID No. 2, and a probe with the nucleotide sequence shown in SEQ ID No. 3, a dPCR premix, and the genomic DNA of the sample to be tested. The cdPCR reaction system, in a total volume of 20 μL, includes: 2.0 μL of 10×dPCRMix; 1.0 μL of the upstream primer shown in SEQ ID No. 1, with a concentration of 10 μmol / L; 1.0 μL of the downstream primer shown in SEQ ID No. 2, with a concentration of 10 μmol / L; 1.0 μL of the probe shown in SEQ ID No. 3, with a concentration of 10 μmol / L; 2.0 μL of the genomic DNA template of the sample to be tested; and the remainder being nuclease-free water.

[0084] S3. Add the cdPCR reaction system to the cdPCR chip, seal the slide and amplify it using the instrument. Incubate at 50℃ for 10 minutes; pre-denature at 95℃ for 5 minutes; then perform 45 cycles of amplification, each cycle including denaturation at 95℃ for 30 seconds and annealing / extension at 56℃ for 45 seconds; finally store at 4℃.

[0085] S4. Use a chip reader to read the amplified fluorescence signal and calculate the absolute copy number of large yellow croaker iridovirus in the test sample based on the number of positive droplets.

[0086] Table 2 shows the detection results of cdPCR and traditional qPCR methods on clinical samples.

[0087] As shown in Table 2, traditional qPCR detection methods can only provide qualitative results (positive / negative) based on the cycle threshold (Ct value), but cannot perform absolute quantification. Therefore, the specific concentration of the virus in the sample cannot be directly determined from the detection result. In contrast, the cdPCR method used in this invention directly provides the absolute copy number (copies / μL) of the large yellow croaker iridovirus nucleic acid, achieving absolute quantification and providing precise quantitative data that qPCR cannot offer. Furthermore, all samples with cdPCR results of 0 copies / μL (11 samples in total) had corresponding qPCR results of "negative"; all samples with cdPCR results >0 copies / μL (10 samples in total) had corresponding qPCR results of "positive". This demonstrates perfect consistency between the two methods, verifying the excellent specificity of the cdPCR method of this invention and the absence of false positives or false negatives. Secondly, this result strongly proves that this invention, while providing quantitative information, fully inherits the reliable qualitative judgment capability of the traditional qPCR method, ensuring the accuracy of the diagnostic results.

[0088] In summary, the primer combinations, kits, and cdPCR detection methods provided by this invention have the following outstanding technical effects:

[0089] 1) High specificity: It has high specificity for the LYCIV MCP gene and no cross-reaction with common aquatic pathogens such as NNV and DIV1;

[0090] 2) High sensitivity: The detection limit can reach 6.2 copies / μL, making it suitable for early detection of extremely low viral loads;

[0091] 3) Good repeatability: The coefficients of variation for both intra-batch and inter-batch repeatability are less than 8.1%, indicating stable and reliable results;

[0092] 4) Absolute quantification: Based on cdPCR technology, the viral copy number can be directly calculated without a standard curve;

[0093] 5) Easy to operate: The reagent kit integrates core components, which facilitates standardized promotion and use.

[0094] Example 7 Primer combination for detecting iridovirus in large yellow croaker

[0095] This embodiment provides a primer composition for detecting iridovirus in large yellow croaker. Example 2 consists of primers and probes designed and prepared. The specific primers include an upstream primer with a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 2. The nucleotide sequence of the probe is shown in SEQ ID No. 3.

[0096] Example 8 Primer combination for detecting iridovirus in large yellow croaker

[0097] The difference from Example 7 is that the primer composition for detecting iridovirus in large yellow croaker in this example preferably has a probe labeled with a fluorescent reporter group VIC at the 5' end and a quenching group BHQ1 at the 3' end.

[0098] Example 9: A kit for cdPCR detection of iridovirus in large yellow croaker

[0099] This embodiment provides a kit for cdPCR detection of iridovirus in large yellow croaker, which contains the primer combination for detecting iridovirus in large yellow croaker provided in Example 7 or 8.

[0100] Example 10: Another kit for cdPCR detection of iridovirus in large yellow croaker

[0101] This embodiment provides another kit for cdPCR detection of large yellow croaker iridovirus. Based on the kit in Example 9, it adds dPCR premix, consumables for making cdPCR chips, and / or a positive control, which is a recombinant plasmid containing the gene sequence of the major capsid protein of large yellow croaker iridovirus.

[0102] In summary, this invention, by combining a highly specific primer-probe system with an advanced chip-based digital PCR technology platform, successfully provides a primer combination, kit, and cdPCR detection method that is highly specific, sensitive, absolutely quantifiable, and reproducible. This method is significantly superior to the existing qPCR method for LYCIV detection, providing strong technical support for the accurate monitoring and effective control of iridovirus disease in large yellow croaker.

[0103] Finally, it should be noted that although the above embodiments have been described in the text and drawings of this application, this should not limit the scope of patent protection of this application. Any technical solutions that are based on the essential concept of this application and utilize the content described in the text and drawings of this application, resulting in equivalent structural or procedural substitutions or modifications, as well as the direct or indirect application of the technical solutions of the above embodiments to other related technical fields, are all included within the scope of patent protection of this application.

Claims

1. Primer combination for detecting iridovirus in large yellow croaker, characterized in that, The primer combination for detecting iridovirus in large yellow croaker includes specific primers and probes. The specific primers include an upstream primer with a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No.

2. The nucleotide sequence of the probe is shown in SEQ ID No.

3.

2. The primer combination for detecting iridovirus in large yellow croaker according to claim 1, characterized in that, The probe is labeled with a fluorescent reporter group at its 5' end and a quencher group at its 3' end. Preferably, the fluorescent reporter group is VIC and the quencher group is BHQ1.

3. A kit for detecting iridovirus in large yellow croaker using cdPCR, characterized in that, It includes the primer combination as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, Also includes: dPCR premix, consumables for making cdPCR chips, and / or positive control products; Preferably, the positive control is a recombinant plasmid containing the LYCIV MCP gene, the major capsid protein gene of large yellow croaker iridovirus.

5. A method for detecting iridovirus in large yellow croaker using cdPCR, characterized in that, Includes the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Prepare a cdPCR reaction system, wherein the cdPCR reaction system comprises the primer combination as described in claim 1 or 2, the dPCR premix, and the genomic DNA of the sample to be tested; S3. Add the cdPCR reaction system to the cdPCR chip, seal the slide, and amplify it using a microarray. S4. Use a chip reader to read the amplified fluorescence signal and calculate the absolute copy number of large yellow croaker iridovirus in the test sample based on the number of positive droplets.

6. The method according to claim 5, characterized in that, The cdPCR reaction system described in step S2, in a total volume of 20 μL, comprises: 10×dPCRMix 2.0 μL; For example, 1.0 μL of the upstream primer shown in SEQ ID NO.1 has a concentration of 10 μmol / L; For example, 1.0 μL of the downstream primer shown in SEQ ID NO.2 has a concentration of 10 μmol / L; The probe shown in SEQ ID NO.3 is 1.0 μL, with a concentration of 10 μmol / L; The genomic DNA template of the sample to be tested was 2.0 μL. The remainder is nuclease-free water.

7. The method according to claim 5, characterized in that, The amplification procedure described in step S3 is as follows: Incubate at 50°C for 10 minutes; pre-denature at 95°C for 5 minutes; then perform 45 cycles of amplification, each cycle including denaturation at 95°C for 30 seconds and annealing / extension at 56°C for 45 seconds; finally store at 4°C.

8. The method according to claim 5, characterized in that, The coefficients of variation for both intra-batch and inter-batch repeatability tests were less than 8.1%.

9. The method according to claim 5, characterized in that, Its detection limit is 6.2 copies / μL.

10. The use of the primer combination as described in claim 1 or 2, or the kit as described in claim 3 or 4, in the preparation of a product for diagnosing iridovirus disease in large yellow croaker.

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