Primer probe group, kit and method for detecting koi herpesvirus based on fluorescent RAA

Abstract

The invention discloses a primer probe group, a kit and a method for detecting koi herpesvirus based on fluorescent RAA. According to the invention, specific primer pairs (SEQ ID NO.5-6) and probes (SEQ ID NO.11) are designed and screened according to a conserved sequence of a KHV (Koi Herpesvirus) TK gene, and a recombinase-mediated isothermal amplification (RAA) fluorescence detection method of KHV is established. The method has the advantages of high detection speed, simplicity in operation, high sensitivity, good specificity and the like, provides an effective technical means for rapid screening of on-site pathogens, and can meet the epidemic disease monitoring requirements of the production line.

Description

Technical Field

[0001] This invention belongs to the field of virus detection, and specifically relates to a primer and probe set, kit and method for detecting koi herpesvirus based on fluorescent RAA. Background Technology

[0002] Koi herpesvirus (KHV), also known as Cyprinidherpesvirus 3 (CyHV-3), belongs to the family Alloherpesviridae and the genus Cyprinivirus. It is the pathogen that causes herpesvirus disease (KHVD) in cyprinid fish. KHV is a double-stranded DNA virus, icosahedral in shape (nucleocapsid T=16 symmetry), with an envelope and an outer membrane. The size of the virus particle varies depending on the type of cell it infects, ranging from 170 to 230 nm in diameter. The genome is approximately 295 kbp long and contains 164 open reading frames (ORFs), of which 156 encode unique proteins. KHV is a highly pathogenic and infectious microorganism. Carp (Cyprinus carpio), koi (Cyprinus carpio haematopterus), and their hybrid offspring are susceptible, while goldfish (Carassius auratus), sturgeon (Acipenser sinensis), and plankton in aquatic bodies can serve as potential carriers. The incubation period for KHV infection is 7–15 days, with a mortality rate as high as 80–100%. KHV can survive for 21 hours in natural water at 23°C, and for 3 days or even longer at the bottom of ponds at 15°C. It remains active even in ice water at 3°C. In the late 1990s, Germany experienced its first large-scale outbreak of KHVD, resulting in the deaths of a large number of koi and carp, which subsequently spread globally. Due to its transmissibility and high pathogenicity, the World Organisation for Animal Health (WOAH) has listed KHVD as a reportable animal disease and it is also a key disease of concern in international trade in aquatic animals.

[0003] The key to fish disease prevention and control lies in early, rapid, and accurate detection. Traditional methods for detecting KHV include cell culture isolation, polymerase chain reaction (PCR), nested PCR, real-time quantitative PCR, and loop-mediated isothermal amplification (LAMP). However, these methods have drawbacks such as long detection time, cumbersome operation, and high requirements for equipment and personnel, which are not conducive to rapid detection of samples in the field.

[0004] Recombinase-aided amplification (RAA) is a convenient and rapid detection method that has emerged in recent years and has been widely used in aquatic disease research. This method mainly utilizes recombinant proteases and single-stranded binding proteins in the reaction, eliminating the temperature cycling process required in conventional PCR. Combined with fluorescent probes, it can achieve rapid and visual detection in as little as 5 minutes, exhibiting high sensitivity, strong specificity, simple operation, and low requirements for reaction conditions. Therefore, developing a specific, sensitive, and convenient real-time fluorescent RAA method for detecting KHVD can provide new technical support for the prevention and control of KHVD. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a primer and probe set for detecting koi herpesvirus based on fluorescent RAA.

[0006] Another objective of this invention is to provide a kit for detecting koi herpesvirus based on fluorescent RAA.

[0007] Another object of the present invention is to provide a method for detecting koi herpesvirus based on fluorescent RAA.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A primer-probe set for detecting koi herpesvirus based on fluorescent RAA consists of a pair of RAA primers and a single RAA probe, the nucleotide sequences of which are as follows:

[0010] 5F: 5′-GCGGCCGGTGGGTTTCTGCTTCTTGGGTTTGGGA-3′ (SEQ ID NO. 5);

[0011] 1R: 5′-CTGGCCTCGGAGAGCATGACGGCGATGGAGTTGGG-3′ (SEQ ID NO. 6);

[0012] KHV-Probe: 5′-GAAACTGGAACTGTCTGATGAGCGTGGGG[FAM-dT]CA[THF]AG[BHQ1-dT]TGCACATGGGCA-3′C3 spacer (SEQ ID NO.11); where “FAM-dT” indicates that the fluorescent group replaces the T base at this position; “THF” indicates tetrahydrofuran; “BHQ1-dT” indicates that the fluorescent group of the black hole quencher BHQ1 replaces the T base at this position; and “C3 spacer” indicates the polymerase extension blocking group C3 spacer.

[0013] A kit for detecting koi herpesvirus based on fluorescent RAA, comprising the aforementioned primer and probe set for detecting koi herpesvirus based on fluorescent RAA.

[0014] In the kit, the concentration of RAA primers is 5–50 μmol / L (preferably 10–30 μmol / L; more preferably 15 μmol / L), and the concentration of probes is 2–30 μmol / L (preferably 10–25 μmol / L; more preferably 15 μmol / L).

[0015] The kit further includes at least one of a positive control and a negative control.

[0016] The application of the primer-probe set or kit for detecting koi herpesvirus based on fluorescent RAA in the detection of koi herpesvirus for purposes other than disease diagnosis and treatment.

[0017] A method for detecting koi herpesvirus based on fluorescent RAA for non-disease diagnostic and therapeutic purposes includes the following steps:

[0018] (1) Nucleic acid extraction

[0019] Nucleic acid is extracted from the sample to be tested and used as a nucleic acid template;

[0020] (2) RAA amplification

[0021] The RAA amplification system (50 μL) was prepared as follows: 25 μL A Buffer, 2 μL each of 10 μmol / L RAA forward and reverse primers, 0.6 μL 10 μmol / L RAA probe, 5 μL DNA template, 12.9 μL ddH2O, and 2.5 μL B Buffer. RAA amplification was then performed using a real-time fluorescence PCR instrument at 37–41 °C to obtain the RAA amplification product. The nucleotide sequences of the RAA forward and reverse primers are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively, and the nucleotide sequence of the RAA probe is shown in SEQ ID NO. 11, respectively.

[0022] (3) Result judgment

[0023] If the test result in step (2) shows a fluorescent signal and gradually increases, it indicates that the sample to be tested is a positive sample for koi herpesvirus (containing koi herpesvirus); conversely, if the test result in step (2) shows no obvious fluorescent signal, it indicates that the sample to be tested is a negative sample for koi herpesvirus.

[0024] The preferred temperature for RAA amplification in step (2) is 39°C.

[0025] The RAA amplification time in step (2) is 20 to 40 minutes; preferably 20 minutes.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] (1) This invention establishes a rapid, sensitive and point-of-care testing (POCT) method for detecting koi herpesvirus (KHV): based on the conserved sequence of the KHV TK gene, specific primers and probes are designed, the best primer pairs are screened, and a recombinase-mediated isothermal amplification (RAA) fluorescence detection method for KHV is established.

[0028] (2) The lowest detection limit of the method established in this invention is 7.01 × 10⁻⁶. 2 The number of copies / μL is consistent with the WOAH-recommended PCR detection method for the TK gene (both are 7.01 × 10⁻⁶). 2 The number of copies / μL was higher than that of the Sph gene PCR detection method (7.01×10⁻⁶ copies / μL). 3 The RAA fluorescence method has good specificity and shows no cross-reactivity with carp spring viremia virus (SVCV), red sea bream iridovirus (RSIV), goldfish hematopoietic organ necrosis virus (CyHv-2), eel herpesvirus (AngHv-1), and viral neuronecrosis virus (VNNV). The results of clinical sample testing are consistent with the two testing methods recommended by WOAH, and only require 20 minutes.

[0029] (3) The koi herpesvirus RAA fluorescence detection method established in this invention has the advantages of fast detection speed, simple operation, high sensitivity and good specificity. The fluorescence RAA method only requires a simple, portable and inexpensive constant temperature fluorescence instrument to complete the detection. It can provide an effective technical means for rapid screening of pathogens in farms with limited laboratory conditions and customs supervision sites, and can meet the disease monitoring needs of the production line. Attached Figure Description

[0030] Figure 1 The results are shown in the primer screening diagram. A represents the fluorescence values ​​after reactions with different downstream primers R (in the diagram, 1: 1F / 1R; 2: 1F / 2R; 3: 1F / 3R; 4: 1F / 4R; 5: 1F / 5R); B is a statistical graph of fluorescence values ​​and CT values ​​after reactions with different downstream primers R (error bars represent mean ± standard deviation, where n = 3 replicates); C represents the fluorescence values ​​after reactions with different upstream primers F (in the diagram, 1: 1R / 1F; 2: 1R / 2F; 3: 1R / 3F; 4: 1R / 4F; 5: 1R / 5F); D is a statistical graph of fluorescence values ​​and CT values ​​after reactions with different upstream primers F (error bars represent mean ± standard deviation, where n = 3 replicates).

[0031] Figure 2This is a graph showing the results of primer concentration screening; where A represents the fluorescence values ​​after reacting with primers of different concentrations (in the graph, 1: 50 μmol / L; 2: 30 μmol / L; 3: 20 μmol / L; 4: 15 μmol / L; 5: 10 μmol / L; 6: 5 μmol / L); and B is a statistical graph of fluorescence values ​​and CT values ​​after reacting with primers of different concentrations (error bars represent the mean ± standard deviation, where n = 3 replicates).

[0032] Figure 3 The graph shows the results of probe concentration screening. In the graph, A represents the fluorescence values ​​after reacting with probes of different concentrations (1: 25 μmol / L; 2: 20 μmol / L; 3: 15 μmol / L; 4: 10 μmol / L; 5: 5 μmol / L; 6: 2 μmol / L); B is a statistical graph of fluorescence values ​​and CT values ​​after reacting with probes of different concentrations (error bars represent the mean ± standard deviation, where n = 3 replicates).

[0033] Figure 4 The results of the reaction temperature screening are shown in the figure; where A is the fluorescence value after the reaction at different temperatures (in the figure, 1: 41℃; 2: 40℃; 3: 39℃; 4: 38℃; 5: 37℃); B is a statistical graph of fluorescence value and CT value after the reaction at different temperatures (error bars represent the mean ± standard deviation, where n = 3 replicates).

[0034] Figure 5 This is a graph showing the sensitivity test results of the fluorescent PCR method (in the graph, 1–7: KHV standard plasmid copy number 7.01 × 10⁻⁶). 6 ~7.01×10 0 copies / μL; 8: negative control).

[0035] Figure 6 This is a graph showing the sensitivity test results of the RAA fluorescence method (in the graph, 1-7: KHV standard plasmid copy number 7.01×10⁻⁶). 6 ~7.60×10 0 copies / μL; N: negative control; **: P<0.01, ns: no significant difference; error bar represents mean ± standard deviation, where n = 3 replicates); where A to C are the results of three replicate tests respectively; D is a statistical graph of fluorescence value and CT value.

[0036] Figure 7 This is a graph showing the results of a routine PCR test; where A represents the PCR test results for the TK gene (in the graph, 1–10: KHV standard plasmid copy number 7.01 × 10⁻⁶). 9 ~7.60×10 0copies / μL; N: negative control); B represents the Sph gene PCR detection results (in the figure, 1–10: KHV standard plasmid copy number 7.01 × 10⁻⁶). 9 ~7.60×10 0 copies / μL; N: negative control).

[0037] Figure 8 This is a graph showing the specificity test results of the RAA fluorescence method (in the graph, 1: positive control (7.01×10⁻⁶)). 5 2: KHV; 3-7: SVCV, RSIV, CyHv-2, AngHv-1, VNNV; 8: Negative control.

[0038] Figure 9 The figure shows the stability test results of the RAA fluorescence method (in the figure, 1: 7.01×10). 5 copies / μL; 2: 7.01×10 4 copies / μL; 3: 7.01×10 3 copies / μL; 4: 7.01×10 2 copies / μL; error bars represent mean ± standard deviation, n = 3 replicates); where A is the concentration of 7.01 × 10⁻⁶. 5 copies / μL and 7.01×10 4 Results of three repeated tests with copies / μL; B is 7.01×10 3 copies / μL and 7.01×10 2 Results of three repeated tests per copies / μL; C is a statistical graph of fluorescence intensity versus CT value. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0040] Example 1

[0041] 1. Materials and Methods

[0042] 1.1 Materials and Equipment

[0043] Samples of carp spring viremia virus (SVCV), red sea bream iridovirus (RSIV), goldfish hematopoietic organ necrosis virus (CyHv-2), eel herpesvirus (AngHv-1), and viral neuronecrosis virus (VNNV) were preserved in the Animal Quarantine Laboratory of the Gongbei Customs Technology Center and were all confirmed by sequencing. Koi herpesvirus (KHV) cell culture medium (inactivated) came from the Shenzhen Customs Animal and Plant Quarantine Technology Center and was confirmed by sequencing.

[0044] The DNA isothermal rapid amplification kit (fluorescent type) and Genchek real-time fluorescence detector were purchased from Hangzhou Zhongce Biotechnology Co., Ltd.; the viral DNA / RNA extraction kit (4.0) and the fully automated rotary nucleic acid extractor GeneRotex were purchased from Xi'an Tianlong Technology Co., Ltd.; the QuantStudio 5 real-time fluorescence PCR instrument was purchased from Applied Biosystems, USA; the 5424R high-speed refrigerated centrifuge was purchased from Eppendorf AG, Germany; and the ND-1000 Spectrohotometer was purchased from NanoDrop Technologies, USA.

[0045] 1.2 Virus preparation and nucleic acid extraction

[0046] The viruses (SVCV, RSIV, CyHv-2, AngHv-1, VNNV, KHV) and clinical test samples used in this experiment were all extracted for nucleic acid according to the instructions of the Tianlong DNA / RNA Nucleic Acid Extraction Kit. The extracted nucleic acid was detected using a UV spectrophotometer. The template A260 / A280 ratio was required to be between 1.8 and 2.0. The samples were stored at -20℃ for later use.

[0047] 1.3 Primer Design and Synthesis

[0048] Based on the KHV isolate gene information published in GenBank, and after comparison using DNAMAN software, the TK gene with accession number MK260013.1 was selected as the target. Following the RAA primer and probe design principles, five pairs of primers and one probe were designed using the biological software Oligo7. The probe carries the antigen marker FAM at its 5′ end (30 nt), which is linked to the 3′ end via the tetrahydrofuran (THF) site. The 3′ end (15 nt) is extended with a polymerase-based blocking group (C3 spacer). Both primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their sequence information is shown in Table 1.

[0049] Table 1. Probe and primer sequences for RAA

[0050]

[0051] Note: In Table 1, “FAM-dT” means that the fluorescent group replaces the T base at this position; “THF” means tetrahydrofuran; “BHQ1-dT” means that the fluorescent group of the black hole quencher BHQ1 replaces the T base at this position; “C3 spacer” means polymerase extension blocking group C3 spacer.

[0052] Construction of 1.4KHV plasmid standards

[0053] In MK260013.1, primers were selected to detect the desired sequence region. A KHV-positive plasmid was synthesized by BGI Genomics Co., Ltd. and ligated into the PUC57 vector to obtain the recombinant plasmid. The total nucleic acid mass of the recombinant plasmid was then extracted to 5 μg, and the copy number was calculated using the formula: Copy number (copies / μL) = (6.02 × 10⁻⁶). 23 The recombinant plasmid was serially diluted 10-fold (copies / μL) × (concentration g / μL) / (molecular mass g / mol) for later use.

[0054] 1.5 Establishment of the RAA reaction system and optimization of reaction conditions

[0055] 1.5.1 RAA primer set screening and reaction system establishment

[0056] First, the designed upstream primer 1F was paired with five downstream primers (1R to 5R) to construct reaction systems. The optimal downstream primer was selected based on fluorescence value and reaction time. Then, the selected downstream primer was paired with the five upstream primers (1F to 5F) to construct reaction systems, ultimately obtaining the optimal upstream and downstream primer combinations. Primer selection was performed according to the instructions of the DNA Isothermal Rapid Amplification Kit (fluorescent type). Each reaction unit contained 25 μL of A Buffer, 2 μL each of 10 μmol / L upstream and downstream primers, 0.6 μL of 10 μmol / L probe, 5 μL of DNA template, 12.9 μL of ddH2O, and 2.5 μL of B Buffer, for a total volume of 50 μL. After mixing, RAA amplification was performed using a QuantStudio 5 real-time fluorescence PCR instrument at 39℃.

[0057] 1.5.2 Optimization of RAA Reaction Conditions

[0058] First, the probe concentration was set at 10 μmol / L. Using the optimal primer combination screened in section 1.5.1, the upstream and downstream primers were prepared at concentrations of 5, 10, 15, 20, 30, and 50 μmol / L (the kit recommends a concentration of 10 μmol / L) (the upstream and downstream primer concentrations were the same during amplification) to screen for optimal primer concentrations. After determining the optimal primer concentration, the probe concentration was prepared at 30, 20, 15, 10, 5, and 2 μmol / L to screen for optimal probe concentration. After determining the optimal primer and probe concentrations, the reaction temperature was set at 37, 38, 39, 40, and 41℃ to test the RAA reaction temperature (the kit recommends an optimal reaction temperature of 39℃) to determine the optimal reaction temperature.

[0059] 1.6 Sensitivity Test

[0060] The recombinant plasmid standard from section 1.4 above, diluted at different ratios, was used as a template (copy number 7.01 × 10⁻⁶). 9 ~7.01×10 0 The sensitivity of the RAA fluorescence detection method was tested using copies / μL, and compared with the fluorescence PCR detection method in Chapter 2.3.6, Section 4.4.2, and Section 4.4.3, Conventional PCR, of the WOAH Manual for the Diagnosis of Aquatic Animal Diseases (2022 Edition). ddH2O was used as a negative control. The primers and probe sequences used for both fluorescence PCR and conventional PCR (targeting the TK or Sph gene) were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the sequence information is shown in Table 2.

[0061] Table 2 Primer and probe sequences

[0062]

[0063]

[0064] Note: In Table 2, "FAM" indicates FAM fluorescent dye labeling; "TAMRA" indicates TAMRA fluorescent dye labeling.

[0065] 1.7 Specificity Test

[0066] Using the nucleic acids of SVCV, RSIV, CyHv-2, AngHv-1, VNNV, and KHV extracted in 1.2 above as templates, the RAA fluorescence method optimized in 1.5 above was used for the experiment. The KHV plasmid standard was used as a positive control, and the DNA sample extracted from healthy carp tissue was used as a negative control.

[0067] 1.8 Repeatability Test

[0068] Take 7.01×10 5copies / μL~7.01×10 2 Four concentrations of KHV standard plasmids (copies / μL) were used for fluorescent RAA reaction, with each concentration repeated three times to evaluate the stability of the detection method.

[0069] 1.9 Clinical Sample Trials

[0070] Twenty freshwater fish (carp) samples and one KHV-positive cell culture medium (inactivated) from surrounding aquaculture farms were collected. Nucleic acid was extracted from the samples using a GeneRotex fully automated rotary nucleic acid extractor. The RAA fluorescence method established in this study and two detection methods (fluorescent PCR and conventional PCR) recommended in the WOAH Manual for the Diagnosis of Aquatic Animal Diseases were used simultaneously for testing, and the results were compared.

[0071] 2 Results and Analysis

[0072] 2.1 Screening results of RAA primer sets

[0073] Following the primer combination screening method in 1.5.1, the experiment was repeated three times. The results showed that the 1F / 1R combination exhibited the highest fluorescence value, the lowest CT value, and the lowest SD. Figure 1 A and B in the middle, Figure 1 The primers marked F1 / R1 were used to select 1R as the downstream primer; then, upstream primers were screened using 1R, and the results showed that 1R / 5F produced the highest fluorescence value, the lowest CT value, and the lowest SD. Figure 1 C and D in the middle, Figure 1 (The text indicates R1 / F5). Based on the combined experimental results, 5F / 1R was determined to be the optimal primer combination, and this primer combination was used for subsequent experiments.

[0074] 2.2 Optimization of Reaction Conditions

[0075] 2.2.1 Screening of primer concentrations

[0076] Using the optimal primer combination (5F / 1R) selected in section 2.1, three tests were conducted with different primer concentrations. The results showed that amplification efficiency was optimal at a primer concentration of 15 μmol / L; this concentration was determined as the optimal primer concentration for subsequent experiments. Figure 2 ).

[0077] 2.2.2 Screening of probe concentration

[0078] Using the optimal primer concentration (15 μmol / L) selected in section 2.2.1, reactions were carried out with probes of different concentrations, with each reaction repeated three times. The results showed no significant differences in fluorescence intensity and CT value at probe concentrations of 10 μmol / L, 15 μmol / L, 20 μmol / L, and 25 μmol / L, but the fluorescence and CT values ​​fluctuated least at a probe concentration of 15 μmol / L. Figure 3 Therefore, the optimal probe concentration was determined to be 15 μmol / L for subsequent experiments.

[0079] 2.2.3 Screening of reaction temperature

[0080] Using the optimal primer and probe concentrations screened in sections 2.2.1 and 2.2.2, tests were conducted at different reaction temperatures, with each reaction repeated three times. The results showed that the fluorescence value was highest at a reaction temperature of 39℃, with a lower and more stable CT value. Figure 4 ), and determined that 39℃ was the optimal reaction temperature for subsequent experiments.

[0081] 2.3 Sensitivity Test

[0082] Based on the copy number calculation formula, the copy number of the KHV standard plasmid is 7.01 × 10⁻⁶. 9 Copies / μL, using standard plasmids of different concentration gradients as templates, and applying fluorescent PCR methods respectively ( Figure 5 ), RAA fluorescence method ( Figure 6 ) and conventional PCR methods ( Figure 7 Sensitivity tests were performed, with each method tested in triplicate. Results showed that the fluorescence values ​​of both fluorescence detection methods decreased with decreasing template concentration, and the detection limit for the fluorescent PCR method was 7.01 × 10⁻⁶. 1 copies / μL; fluorescence RAA method at a concentration of 7.01×10 2 At 7.01 × 10⁻⁶ copies / μL, the fluorescence value was significantly different from that of the negative control group (ddH₂O) (P < 0.01), while at 7.01 × 10⁻⁶... 1 The fluorescence values ​​detected at copies / μL were not significantly different from those of the negative control group (ddH2O), thus determining the detection limit of the RAA fluorescence method to be 7.01 × 10⁻⁶. 2 The limit of detection for the TK gene PCR assay (Bercovier et al.) is 7.01 × 10⁻⁶ copies / μL; 2 copies / μL ( Figure 7 In section A), the detection limit of the PCR detection method for Sph gene (Serine protease inhibitor genes) (Gray et al.) is 7.01 × 10⁻⁶. 3copies / μL ( Figure 7 (B in the middle).

[0083] 2.4 Specificity test

[0084] The established RAA fluorescence detection method was used to test the nucleic acids of positive samples of SVCV, RSIV, CyHv-2, AngHv-1, VNNV, and KHV, with each reaction repeated three times. Results showed that KHV nucleic acid exhibited an amplification curve, while negative samples and other viral nucleic acid tests were all negative. Figure 8 This indicates that the established RAA fluorescence method has good specificity.

[0085] 2.5 Repeatability Test

[0086] With 7.01×10 5 copies / μL~7.01×10 2 Using KHV standard plasmids at four concentrations (copies / μL) as templates, three intra-group repeatability tests were performed for each concentration. The results showed that the fluorescence peaks at each concentration were similar, and good amplification curves were observed, indicating that the established method has good repeatability. Figure 9 ).

[0087] 2.6 Detection of clinical samples

[0088] The RAA fluorescence method established in this experiment, along with the fluorescence PCR and PCR methods recommended in the WOAH Manual for the Diagnosis of Aquatic Animal Diseases (targeting the TK gene, primer sequences are shown in Table 2), were used to test 20 freshwater fish samples and 1 confirmed positive sample submitted by Gongbei Customs District. Each reaction was repeated three times. The results showed that the three detection methods yielded consistent results, while the RAA fluorescence detection method established in this study had the shortest detection time, taking only 20 minutes.

[0089] Table 3 Comparison of Clinical Sample Detection Results

[0090]

[0091] 3 Discussion

[0092] This study designed specific primers and probes targeting the KHV TK gene sequence to establish a fluorescent detection method for koi herpesvirus (KHV) RAA. Sensitivity, specificity, and clinical sample test results showed that the sensitivity of the established method was slightly lower than the fluorescent PCR method recommended in the WOAH Manual for the Diagnosis of Aquatic Animal Diseases, higher than the conventional PCR detection method for the Sph gene, and consistent with the sensitivity of the conventional PCR detection method for the TK gene. The fluorescent RAA detection method showed good specificity and no cross-reactivity with other aquatic animal pathogens. Intra-group repeatability tests indicated good stability of the detection method. Clinical multi-sample test results were consistent with the two detection methods recommended by WOAH. Regarding detection efficiency, since the RAA reaction principle is based on the recombinase-mediated action, the repeated thermal cycling process is omitted, achieving nucleic acid amplification in an isothermal environment (39℃). Therefore, the entire reaction time is only 20 minutes, and only a portable isothermal fluorescence detector is required.

[0093] In summary, the RAA fluorescence detection method for koi herpesvirus established in this study is characterized by high sensitivity, strong specificity, good stability, and simplicity and speed. It can be used for rapid detection and screening of suspected infected samples and for KHVD monitoring. Combined with a simple nucleic acid extraction method, it can achieve true point-of-care testing (POCT). This method meets the needs of rapid pathogen screening in aquaculture farms with limited laboratory conditions and at customs supervision sites. It is of great significance for improving the early warning and control capabilities of foreign diseases, real-time monitoring of KHVD transmission, protecting the healthy and stable development of agriculture and fisheries, and facilitating international trade.

[0094] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A primer and probe set for detecting koi herpesvirus based on fluorescent RAA, characterized in that, It includes a pair of RAA primers and one RAA probe, with the following nucleotide sequence: 5F: 5′-GCGGCCGGTGGGTTTCTGCTTCTTGGGTTTGGGA-3′ (SEQ ID NO. 5); 1R: 5′-CTGGCCTCGGAGAGCATGACGGCGATGGAGTTGGG-3′ (SEQ ID NO. 6); KHV-Probe: 5′-GAAACTGGAACTGTCTGATGAGCGTGGGG[FAM-dT]CA[THF]AG[BH Q1-dT]TGCACATGGGCA-3′C3 spacer (SEQ ID NO. 11).

2. A kit for detecting koi herpesvirus based on fluorescent RAA, characterized in that, Includes the primer and probe set for detecting koi herpesvirus based on fluorescent RAA as described in claim 1.

3. The reagent kit according to claim 2, characterized in that: In the kit, the concentration of RAA primers is 5–50 μmol / L, and the concentration of probes is 2–30 μmol / L.

4. The reagent kit according to claim 3, characterized in that: In the kit, the concentration of RAA primers is 10–30 μmol / L, and the concentration of probes is 10–25 μmol / L.

5. The reagent kit according to claim 4, characterized in that: In the kit, the concentration of RAA primers is 15 μmol / L, and the concentration of probes is 15 μmol / L.

6. The application of the primer and probe set for detecting koi herpesvirus based on fluorescent RAA as described in claim 1 or the kit for detecting koi herpesvirus based on fluorescent RAA as described in any one of claims 2 to 5 in the detection of koi herpesvirus for purposes other than disease diagnosis and treatment.

7. A method for detecting koi herpesvirus based on fluorescent RAA for non-disease diagnosis and treatment purposes, characterized in that, Includes the following steps: (1) Nucleic acid extraction Nucleic acid is extracted from the sample to be tested and used as a nucleic acid template; (2) RAA amplification Prepare the RAA amplification system as follows: Buffer A 25 μL, 10 μmol / L RAA forward and reverse primers 2 μL each, 10 μmol / L RAA probe 0.6 μL, DNA template 5 μL, ddH2O 12.9 μL, Buffer B 2.5 μL; then perform RAA amplification using a real-time fluorescence PCR instrument at 37–41℃ to obtain RAA amplification products; the nucleotide sequences of the RAA forward and reverse primers are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively, and the nucleotide sequence of the RAA probe is shown in SEQ ID NO.11, respectively. (3) Result judgment If the test results in step (2) show a fluorescent signal and gradually increase, it indicates that the sample to be tested is a positive sample for koi herpesvirus; otherwise, if the test results in step (2) show no obvious fluorescent signal, it indicates that the sample to be tested is a negative sample for koi herpesvirus.

8. The method according to claim 7, characterized in that: The RAA amplification temperature described in step (2) is 39°C.

9. The method according to claim 7, characterized in that: The RAA amplification time in step (2) is 20 to 40 minutes.

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