Composition and kit for detecting pathogenic microorganisms of fish and application of composition and kit

Abstract

The invention relates to the technical field of molecular biological detection, and discloses a composition and a kit for detecting pathogenic microorganisms of fish and application of the composition and the kit. The composition provided by the invention comprises a primer pair aiming at a target sequence of at least one of infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, flavobacterium psychrophilum, aeromonas salmonicida and aeromonas hydrophila; according to the method, multiple fish pathogenic microorganisms in a sample can be detected and confirmed only through one-time PCR, the detection efficiency is greatly improved, the detection cost is reduced, and the method has good specificity, high detection sensitivity, convenience in operation and excellent stability.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and more specifically to a composition, kit, and application for detecting pathogenic microorganisms in fish. Background Technology

[0002] In aquaculture environments, fish are susceptible to various pathogenic microorganisms, directly leading to a significant decline in the economic benefits for fish farmers. Among known pathogenic microorganisms, infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, *Flavobacterium psychrophilum*, *Aeromonas salmonidae*, and *Aeromonas hydrophila* pose significant risks. The harm caused by these pathogens is particularly pronounced in salmonid fish. Common farmed species such as Atlantic salmon, Pacific salmon, and rainbow trout are highly susceptible to these pathogens. Therefore, timely detection of their presence is of great significance for controlling the spread of fish diseases and ensuring fisheries production.

[0003] However, these pathogenic microorganisms not only proliferate and spread rapidly, but their detection also presents significant technical challenges. Traditional detection methods either suffer from complex procedures and excessively long testing cycles, resulting in results lagging behind the disease's progression; or they lack sufficient sensitivity, making accurate identification difficult in the early stages of infection, preventing farmers from implementing effective control strategies in a timely manner and thus missing the optimal window for disease control. Conventional single-channel PCR methods require multiple separate tests to confirm the disease type, leading to high testing costs. Not only is the detection efficiency low, failing to meet the needs of rapid disease diagnosis, but it is also prone to missed detections when facing mixed infections of multiple pathogens, delaying control efforts. Furthermore, some detection techniques have stringent requirements for laboratory equipment and the professional skills of operators, making them difficult for grassroots farming units to apply.

[0004] Therefore, there is an urgent need to provide methods and related products that can simultaneously detect multiple pathogenic microorganisms in fish and have high detection sensitivity, specificity and accuracy. Summary of the Invention

[0005] The purpose of this invention is to overcome the aforementioned problems in the prior art and to provide a composition, kit, and application thereof for detecting pathogenic microorganisms in fish.

[0006] To achieve the above objectives, the present invention provides a composition for detecting pathogenic microorganisms in fish, the composition comprising at least one of the following primer pairs: Primer pair A: includes an upstream primer and a downstream primer targeting the target sequence of infectious hematopoietic necrosis virus, wherein the target sequence of infectious hematopoietic necrosis virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:22. Primer pair B: includes an upstream primer and a downstream primer targeting the target sequence of infectious pancreatic necrosis virus, wherein the target sequence of infectious pancreatic necrosis virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:23. Primer pair C: includes an upstream primer and a downstream primer targeting the target sequence of viral hemorrhagic septicemia virus, wherein the target sequence of viral hemorrhagic septicemia virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:24. Primer pair D: includes an upstream primer and a downstream primer for the target sequence of Flavobacterium psychrophilum, wherein the target sequence of Flavobacterium psychrophilum has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:25; Primer pair E: includes an upstream primer and a downstream primer targeting the target sequence of Aeromonas salmonicida, wherein the target sequence of Aeromonas salmonicida has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:26; Primer pair F: includes an upstream primer and a downstream primer for the target sequence of Aeromonas hydrophila, wherein the target sequence of Aeromonas hydrophila has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:27.

[0007] A second aspect of the present invention provides a kit comprising the composition described in the first aspect.

[0008] The third aspect of the present invention provides the use of the composition described in the first aspect, or the kit described in the second aspect, in the detection of fish pathogenic microorganisms in samples, particularly in the detection of fish pathogenic microorganisms in salmonid samples.

[0009] A fourth aspect of the present invention provides a method for detecting fish pathogenic microorganisms in a sample, the method comprising performing nucleic acid amplification on the sample to be tested using the composition described in the first aspect or the kit described in the second aspect, wherein the fish pathogenic microorganism is selected from at least one of infectious hematopoietic organ necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, Flavobacterium psychrophilum, Aeromonas salmonidae, and Aeromonas hydrophila.

[0010] The beneficial effects obtained by the present invention through the above technical solution include at least the following: (1) The primer pairs contained in the composition provided by the present invention can amplify the target sequences of different fish pathogenic microorganisms (infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, psychrophilic flavipes, salmonicidal aeromonas and hydrophila) in one detection. Multiple fish pathogenic microorganisms in the sample can be detected and confirmed by PCR in one PCR, which greatly improves the detection efficiency and reduces the detection cost. The combination with the housekeeping gene internal standard can avoid false negatives and make the detection results more reliable.

[0011] (2) The primer pairs (and optional probes) contained in the composition provided by the present invention do not aggregate with each other, and have good specificity, high detection sensitivity, and are easy to operate, making them suitable for large-scale promotion and application.

[0012] (3) The composition provided by the present invention has excellent stability, can be stored for a long time in a suitable storage environment, and can also maintain its activity for a certain period of time in an unsuitable storage environment, thus adapting to the detection requirements of different environments. Attached Figure Description

[0013] Figure 1 This is a graph showing the results of real-time PCR detection of simulated test samples using kit A from Example 1 in Example 2. Figure 2 This is a graph showing the detection results of the specificity test of kit A from Example 1 in Example 2; Figure 3 This is a graph showing the results of real-time quantitative PCR detection of simulated test samples using kit B in Example 2. Figure 4 This is a graph showing the results of quantitative real-time PCR detection of simulated test samples using the control kit in Comparative Example 1. Detailed Implementation

[0014] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0015] In this invention, "identity" refers to the percentage of identical sequences between two sequences. For example, 90% identity between sequence a and sequence b means that 90% of sequence a is identical to sequence b, or 90% of sequence b is identical to sequence a. Differences between the two sequences can be due to nucleotide deletions, additions, or substitutions. For instance, sequence b, obtained by deleting 20 consecutive nucleotides from the 5' end of sequence a (containing 100 nucleotides), has 80% identity with sequence a, while sequence b has 100% identity with sequence a.

[0016] A first aspect of the present invention provides a composition for detecting pathogenic microorganisms in fish, the composition comprising at least one of the following primer pairs: Primer pair A: includes an upstream primer and a downstream primer targeting the target sequence of Infectious Haematopoietic Necrosis Virus (IHNV), wherein the target sequence of IHNV has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:22. Primer pair B: includes an upstream primer and a downstream primer for the target sequence of infectious pancreatic necrosis virus (IPNV), wherein the target sequence of IPNV has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:23. Primer pair C: includes an upstream primer and a downstream primer for the target sequence of viral hemorrhagic septicemia virus (VHS), wherein the target sequence of viral hemorrhagic septicemia virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:24. Primer pair D: includes targets for Flavobacterium psychrophilum ( Flavobacterium psychrophilum The upstream and downstream primers of the target sequence of Flavovirens psychrophilus, wherein the target sequence of Flavovirens psychrophilus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:25; Primer pair E: includes targets against Aeromonas salmonicida ( Aeromonas salmonicida The upstream and downstream primers of the target sequence of Aeromonas salmonidae, wherein the target sequence of Aeromonas salmonidae has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:26; Primer pair F: includes targets for Aeromonas hydrophila ( Aeromonas hydrophila The upstream and downstream primers of the target sequence of Aeromonas hydrophila, wherein the target sequence of Aeromonas hydrophila has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:27.

[0017]

[0018]

[0019]

[0020] Preferably, the nucleotide sequence of the target sequence of the infectious hematopoietic necrosis virus is shown in SEQ ID NO:22.

[0021] Preferably, the nucleotide sequence of the target sequence of infectious pancreatic necrosis virus is shown in SEQ ID NO:23.

[0022] Preferably, the nucleotide sequence of the target sequence of viral hemorrhagic septicemia virus is shown in SEQ ID NO:24.

[0023] Preferably, the nucleotide sequence of the target sequence of Flavobacterium psychrophilum is shown in SEQ ID NO:25.

[0024] Preferably, the nucleotide sequence of the target sequence for killing Aeromonas salmon is shown in SEQ ID NO:26.

[0025] Preferably, the nucleotide sequence of the target sequence of Aeromonas hydrophila is shown in SEQ ID NO:27.

[0026] More preferably, primer pair A includes primers with nucleotide sequences as shown in SEQ ID NO:1-2.

[0027] More preferably, primer pair B comprises primers with nucleotide sequences as shown in SEQ ID NO:4-5.

[0028] More preferably, primer pair C includes primers with nucleotide sequences as shown in SEQ ID NO:7-8 or SEQ ID NO:33-34.

[0029] More preferably, primer pair D includes primers with nucleotide sequences as shown in SEQ ID NO:10-11.

[0030] More preferably, primer pair E includes primers with nucleotide sequences as shown in SEQ ID NO:13-14.

[0031] More preferably, primer pair F includes primers with nucleotide sequences as shown in SEQ ID NO:16-17.

[0032] According to the present invention, preferably, the composition further includes a nucleic acid probe, the nucleic acid probe comprising at least one of the following: Nucleic acid probe A includes probes targeting the target sequence of infectious hematopoietic necrosis virus; Nucleic acid probe B includes probes targeting the target sequence of infectious pancreatic necrosis virus; Nucleic acid probe C includes probes targeting the target sequence of viral hemorrhagic septicemia virus; Nucleic acid probe D includes probes targeting the target sequence of Flavobacterium psychrophilum; Nucleic acid probe E includes probes targeting the target sequence of Aeromonas salmonii; Nucleic acid probe F includes probes targeting the target sequence of Aeromonas hydrophila.

[0033] More preferably, nucleic acid probe A comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:3.

[0034] More preferably, nucleic acid probe B comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:6.

[0035] More preferably, nucleic acid probe C comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:9 or SEQ ID NO:35.

[0036] More preferably, the nucleic acid probe D comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:12.

[0037] More preferably, nucleic acid probe E comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:15.

[0038] More preferably, the nucleic acid probe F comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:18.

[0039] In the composition provided by the present invention, when multiple sets of primers and multiple sets of probes are available for a certain detection target, the specific primers and probes used in combination are more effective. For example, for primer pair C and nucleic acid probe C, it is preferable to use the primer pair shown in SEQ ID NO:7-8 and the nucleic acid probe shown in SEQ ID NO:9 in combination; or, for another example, it is preferable to use the primer pair shown in SEQ ID NO:33-34 and the nucleic acid probe shown in SEQ ID NO:35 in combination.

[0040] According to a preferred embodiment of the present invention, in order to make the entire experimental process more accurate and reliable, the composition may further include a quality control internal standard, a primer pair G for detecting the quality control internal standard, and (optionally) a probe G for detecting the quality control internal standard.

[0041] Internal control standards are gene fragments not found in the fish genome. Adding internal control standards to the biological samples to be tested can further improve the accuracy of detection. For example, internal control standards are involved in the extraction of nucleic acids from pathogenic microorganisms in fish samples and the PCR reaction process. The presence or absence of amplification signals in the test results can determine whether the sample processing and PCR amplification system are normal. While testing the sample, the nucleic acid purification process in the sample can be monitored, eliminating false negatives.

[0042] Those skilled in the art can select suitable gene fragments as quality control internal standards included in the compositions of this invention based on publicly available information about fish genomes and other species genomes, and design suitable primers and probes for the selected gene fragments. For example, housekeeping genes from plants can be used.

[0043] In a particularly preferred embodiment, the Arabidopsis housekeeping gene is selected as an internal control standard. Arabidopsis (… Arabidopsis thaliana (L.) Heynh. Arabidopsis thaliana is a plant, and the nucleic acid sequence of its housekeeping gene is not homologous to that of fish (animal) genomes. When housekeeping genes from plants such as Arabidopsis thaliana are used as quality control internal standards, the primers and probes of the quality control internal standard will not specifically bind to fish genes in the sample during the detection process, fundamentally avoiding false positive results. Furthermore, by adding the quality control internal standard to the sample and performing nucleic acid extraction, purification, and PCR together with the sample, the amplification status of the quality control internal standard in the detection results can be used to determine whether there are false negative results caused by reagents, operation, or other factors. In addition, the housekeeping gene of Arabidopsis thaliana is constitutively and stably expressed in plants; when used as an exogenous internal standard, its copy number remains constant during experiments, accurately reflecting the efficiency of nucleic acid extraction and PCR amplification.

[0044] According to the present invention, preferably, primer pair G comprises an upstream primer and a downstream primer targeting the Arabidopsis housekeeping gene target sequence, wherein the Arabidopsis housekeeping gene target sequence has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:28.

[0045]

[0046] According to the present invention, preferably, primer pair G comprises primers with nucleotide sequences as shown in SEQ ID NO:19-20.

[0047] According to the present invention, the nucleic acid probe G comprises a probe targeting the Arabidopsis housekeeping gene sequence.

[0048] According to the present invention, preferably, the nucleic acid probe G comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:21.

[0049] Preferably, the nucleic acid probe further includes a fluorescent reporter group modified on the nucleic acid probe (e.g., at the 5' end), which may include at least one of AF405, ATTO 425, HEX, FAM, ROX, CY5, CY7 and Quasar705.

[0050] Preferably, the nucleic acid probe further includes a fluorescence quenching group modified on the nucleic acid probe (e.g., at the 3' end), and the fluorescence quenching group may include at least one of BHQ0, BHQ1, BHQ2, and BHQ3.

[0051] In the composition provided by this invention, each primer and probe can be packaged independently or in combination. Since non-specific binding does not occur between the primers and probes in the composition provided by this invention, preferably, to save packaging materials, reduce production and usage costs, and simplify detection operations, the composition is presented in a mixed packaging form.

[0052] A second aspect of the present invention provides a kit comprising the composition described in the first aspect.

[0053] Preferably, the kit further comprises at least one of a buffer, an enzyme, a ribonuclease inhibitor, a magnesium source, and a deoxyribonucleotide triphosphate.

[0054] More preferably, the buffer solution comprises tris-hydroxymethylaminomethane hydrochloride buffer (Tris-HCl buffer).

[0055] More preferably, the enzyme includes at least one of reverse transcriptase, UNG enzyme (also known as UDG enzyme), and DNA polymerase (e.g., Taq enzyme, H-Taq enzyme, etc.).

[0056] In the kit of this invention, the magnesium source refers to the source of Mg provided to the reaction system. 2+ The reagents used are any reagents in the art that can be used to provide Mg ions for nucleic acid amplification systems and are applicable to this invention. For example, water-soluble Mg salts (such as MgCl2) can be used as magnesium sources.

[0057] This invention does not impose any particular restrictions on the source of the deoxyribonucleoside triphosphates used in the kit. They can be self-prepared or formulated products, or commercially available finished products. For example, commercially available dNTPs reagents (containing dATP, dTTP, dGTP, dCTP) and dNTPs(U) reagents (containing dATP, dTTP, dGTP, dCTP, dUTP) can be used.

[0058] More preferably, the kit also includes a positive control and a negative control.

[0059] This invention does not impose any particular restrictions on the specific selection of reagents used as positive and negative controls in the kit, and they can be selected according to conventional techniques in the art. For example, reagents that do not contain the target sequence, such as physiological saline or buffer, can be used as negative controls, as can internal standard gene plasmids; for example, mixed plasmids containing the corresponding specific gene target sequence, DNA or RNA fragments of the target gene, pseudoviruses, etc., can be used as positive controls.

[0060] The third aspect of the present invention provides the use of the composition described in the first aspect, or the kit described in the second aspect, in the detection of pathogenic microorganisms in fish samples.

[0061] According to a preferred embodiment of the present invention, the fish pathogenic microorganism is selected from at least one of infectious hematopoietic organ necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, Flavobacterium psychrophilum, Aeromonas salmonidae, and Aeromonas hydrophila.

[0062] According to the present invention, the composition or kit provided by the present invention can detect samples from any source to determine whether they contain the aforementioned fish pathogenic microorganisms. For example, the samples can be tissue or cell samples obtained from fish, in vitro cultured cells obtained through cell culture, or environmental samples (e.g., water samples from fish farming sites, water samples used for aquaculture during fish transportation or sales, environmental samples obtained from fish storage sites, etc.).

[0063] Since the aforementioned fish pathogens pose the most significant threat to salmonid fish, it is preferred that the application include the detection of fish pathogens in salmonid fish-related samples.

[0064] The composition and kit based on the present invention can simultaneously detect multiple pathogens, including infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, *Flavobacterium psychrophilum*, *Aeromonas salmonicida*, and *Aeromonas hydrophila*. In other words, during detection, only one PCR test (in the same test tube) is needed to simultaneously obtain detection results for two to six pathogens, thereby significantly improving detection efficiency.

[0065] It is understood that the core purpose of the composition or kit provided by this invention is to confirm whether the sample to be tested contains the aforementioned pathogenic microorganisms of fish through a single test. The test results do not depend on the types and quantities of pathogenic microorganisms in the sample. The specific test results can include the following three categories: (1) If the sample does not contain any of the aforementioned fish pathogenic microorganisms, the test result will be negative; (2) If the sample contains only one of the aforementioned fish pathogenic microorganisms, the test result will only correspond to a single positive result for that pathogenic microorganism; (3) If the sample contains two or more of the aforementioned pathogenic microorganisms of fish, the test result corresponds to multiple positive results for multiple pathogenic microorganisms.

[0066] That is, regardless of whether the sample contains all of the aforementioned fish pathogenic microorganisms, or contains one or more of the aforementioned pathogenic microorganisms, or does not contain any of the aforementioned pathogenic microorganisms, the composition or kit of the present invention can achieve the corresponding result determination through a single test.

[0067] The compositions and kits provided by this invention can detect any one of the pathogens, including infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, Flavobacterium psychrophilum, Aeromonas salmonidae, and Aeromonas hydrophila, and can also detect two, three, four, five, or six of them simultaneously.

[0068] A fourth aspect of the present invention provides a method for detecting fish pathogenic microorganisms in a sample, the method comprising performing nucleic acid amplification on the sample to be tested using the composition described in the first aspect or the kit described in the second aspect, wherein the fish pathogenic microorganism is selected from at least one of infectious hematopoietic organ necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, Flavobacterium psychrophilum, Aeromonas salmonidae, and Aeromonas hydrophila.

[0069] Preferably, the method includes: (1) Extract nucleic acid from the sample to be tested; (2) Amplify the nucleic acid extracted in step (1).

[0070] According to the present invention, in step (1), the nucleic acid extraction process can be carried out using conventional nucleic acid extraction reagents and nucleic acid extraction processes in the art, as long as the nucleic acid can be separated from the sample.

[0071] Preferably, the method further includes analyzing the amplification results. In a preferred embodiment, since the probe is modified with fluorescent labeling groups or other markers, qualitative or quantitative detection of fish pathogenic microorganisms can be achieved by detecting the markers through fluorescence detection or other methods.

[0072] Depending on the type of fish pathogenic microorganism, the fluorescent labeling groups modified on the probes of this invention are different. Therefore, when the kit of this invention contains primers and probes for multiple fish pathogenic microorganisms, multiple fish pathogenic microorganisms in a sample can be detected simultaneously through a single nucleic acid amplification. For example, in some preferred embodiments of this invention, the sample to be tested and the primers and probes in the kit for detecting multiple fish pathogenic microorganisms can be added to the same PCR tube for in vitro nucleic acid amplification. Then, according to the fluorescent labeling groups modified on the probes, the types of fish pathogenic microorganisms in the sample can be analyzed through the amplification results of different channels.

[0073] The applications provided in the third aspect and the methods provided in the fourth aspect of this invention can be diagnostic or non-diagnostic. For example, a diagnostic application / method can be to test samples from subjects (including fish, human patients potentially infected by fish, or laboratory animals) using the compositions or kits provided by this invention to determine whether they are infected with fish pathogens, in order to confirm their illness or treatment status. A non-diagnostic application / method can be to test samples from subjects using the compositions or kits provided by this invention to determine whether they are infected, for further research (e.g., drug screening, disease mechanism research, etc.); it can also be used to test samples from non-subject sources (e.g., water samples, food, pharmaceuticals, etc.) using the compositions or kits provided by this invention to determine whether they contain fish pathogens, thereby assessing environmental pollution, the risk of fish pathogen spread, etc., and developing targeted treatment plans.

[0074] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0075] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical or biological reagent / material suppliers, and all reagents are of analytical grade.

[0076] Example 1 This embodiment illustrates the preparation of reagent kit A provided by the present invention.

[0077] 1. Prepare primers and probes Based on the target sequences of each detection target listed in Table 4, Hunan Kangde Biotechnology Co., Ltd. was commissioned to synthesize primers and probes for detecting infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), Flavobacterium psychrophilum, Aeromonas salmonicida, Aeromonas hydrophila, and Arabidopsis thaliana housekeeping genes (specific sequences are shown in Table 1). Each probe is modified with a fluorescent reporter group and a quencher group (specific modified groups are shown in Table 2). In Table 1, R / Y / M / K / S / W / V represent degenerate bases, where R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents C / G, W represents A / T, and V represents G / A / C. The synthesized primers / probes containing degenerate bases are a mixture of primers / probes that use the corresponding bases at the degenerate base positions, and the molar ratio of primers / probes with different sequences in the mixture is 1:1 (for example, the upstream primer of Aeromonas salmonicidae synthesized is a mixture of nucleotide sequences with A and T at position 14 in a molar ratio of 1:1).

[0078] Table 1

[0079] Table 2

[0080] 2. Prepare the unit reaction reagent kit for PCR reaction. Prepare the reagents according to the required quantities for the unit reaction reagent kits (i.e., reagent kits used to test one sample in one PCR reaction) in Table 3. In Table 3, the PCR buffer is PCR buffer (S09) purchased from Hunan Kangde Biotechnology Co., Ltd.; the reagents are packaged separately as "PCR reaction solution" and "enzyme" in the table, that is, the PCR reaction solution is a mixture of the listed components, the enzyme is a mixture of reverse transcriptase (Neoscript® RTase, catalog number E13), H-Taq enzyme and UNG enzyme; DEPC water is ultrapure water that has been treated with DEPC (diethyl pyrocarbonate) and sterilized by high temperature and high pressure.

[0081] Table 3

[0082] 3. Prepare internal standard reagent and control reagent. Negative control: sterile saline.

[0083] Positive control: A mixture of cloned plasmids containing the target sequences of various pathogens (infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, *Flavobacterium psychrophilum*, *Aeromonas salmonicida*, and *Aeromonas hydrophila*), diluted with 0.01% TE-SDS to a final concentration of 1 × 10⁻⁶ for each target sequence. 7 The copy number / mL was used as a positive control.

[0084] Quality control internal standard: The cloned plasmid containing the target sequence of the Arabidopsis housekeeping gene was diluted with 0.01% TE-SDS to a final concentration of 1×10⁻⁶. 6 The copy number / mL is used as an internal standard for quality control.

[0085] The cloning plasmids for positive control and quality control internal standard were prepared by inserting the corresponding target sequence into the pUC57 vector. The specific target sequences are shown in Table 4.

[0086] Table 4

[0087] 4. Prepare the reagent kit According to the target detection amount of a single kit A, combine and package the reagents prepared in steps 1-3.

[0088] Example 2 Mix the PCR reaction solution, enzyme, and water in Table 3 thoroughly, then centrifuge at 2000 rpm for 10 seconds for later use.

[0089] (a) PCR detection The cloned plasmids containing the target sequences of each pathogen synthesized in Example 1 were mixed and diluted with 0.01% TE-SDS to a final concentration of 1×10⁻⁶. 5 The copy number / mL was used as a simulated test sample and detected using kit A prepared according to the example.

[0090] Take 200 μL of simulated test sample, 200 μL of negative control and 200 μL of positive control into 1.5 mL centrifuge tubes respectively, add 20 μL of quality control internal standard to each centrifuge tube, and use the nucleic acid processing and purification reagent (product number: S10015) of Sansure Biotech Co., Ltd. to perform nucleic acid extraction and purification according to its instructions.

[0091] Take 4 μL of the prepared simulated test sample, 4 μL of the prepared negative control, and 4 μL of the prepared positive control and add them to 0.2 mL PCR reaction tubes respectively. Add 16 μL of the centrifuged PCR reaction reagent kit to each tube, mix well, and place in a fully automated medical PCR analyzer (Shanghai Hongshi Medical Technology Co., Ltd., SLAN-48S). Perform PCR reaction according to the reaction conditions in Table 5.

[0092] Table 5

[0093] Detection criteria: Select the corresponding channel for detection according to the fluorescent reporter groups shown in Table 2. If a channel shows a clear S-shaped amplification curve and a Ct value ≤ 38, the target site corresponding to that channel is considered positive; if a channel shows no amplification curve (No Ct) or a Ct value > 38, the target site corresponding to that channel is considered negative. The detection results of simulated test samples are as follows: Figure 1 As shown, all tests on the simulated sample were positive, consistent with the results of the positive control. The negative control results showed that all channels were negative except for the quality control internal standard (CY5 channel), which was positive.

[0094] (ii) Specific detection The method described in Experiment (I) was used to detect pathogenic microorganisms containing other fish pathogens (Aitwer virus, rainbow trout herpesvirus, Aeromonas guinea pig, Aeromonas sobria, Nephrobacter salmonii, etc.). Among these, Aitwer virus, rainbow trout herpesvirus, and Nephrobacter salmonii also infect and harm salmonid fish, thus overlapping with the host range of the target pathogens detected in this invention. Aeromonas guinea pig and Aeromonas sobria both belong to the Aeromonas genus and also infect fish. In traditional detection methods, the presence of these pathogens often leads to false positive results for the target pathogens detected in this invention. PCR results are as follows: Figure 2 As shown in the figure, kit A showed obvious negative detection results for all the above-mentioned pathogenic microorganisms in fish, indicating that kit A has high specificity for the target pathogens.

[0095] (III) Sensitivity Testing Each target sequence was prepared and diluted to 600 copies / mL, 500 copies / mL, and 400 copies / mL, respectively. The simulated test samples were then tested using the method described in Experiment (I). The results are shown in Table 6, presented as a ratio of the number of target sequences detected to the total number of detected sequences (a / b, where a is the number of detected sequences and b is the total number of detected sequences). The detection limit for each target sequence (i.e., the lowest concentration of the target sequence for which the number of detected sequences ≥ 95% of the total number of detected sequences) was determined to be 500 copies / mL.

[0096] Table 6

[0097] (iv) Anti-interference test The simulated test samples were tested using the method in Experiment (I). The difference was that the interfering substances listed in Table 7 were added to the samples (the amount of interfering substances added was 50 μg / mL). The statistical results of the effect test of each channel after adding the interfering substances (expressed as Ct values) are shown in Table 7.

[0098] Table 7

[0099] (v) Stability testing Test sample preparation method: IHNV samples were diluted with TE buffer to a Ct value of approximately 25, and the extracted nucleic acid was used as Sample 1. Sample 1 was diluted 10-fold with TE buffer to become Sample 2, and so on, until Sample 4 was obtained by 10-fold dilution. Each sample was tested twice to test the linear range. Samples 3-4 were tested 8 times before and after each of the following stability experiments.

[0100] (1) High-temperature accelerated experiment: After storing the PCR reaction solution in a 37°C incubator for 24 hours, its performance was tested according to the method in Experiment (I) (referred to as "after acceleration"). At the same time, the performance of the freshly prepared PCR reaction solution in the control group was tested (referred to as "before acceleration"). The CT values ​​are shown in Table 8.

[0101] Table 8

[0102] (2) Long-term storage experiment: After the PCR reaction solution was stored under actual storage conditions (-20±5℃) for 11 months, its performance was tested according to the method in Experiment (I) (referred to as "after storage"). At the same time, the performance of the newly prepared PCR reaction solution of the control group was tested (referred to as "before storage"). The CT values ​​are shown in Table 9.

[0103] Table 9

[0104] (3) Freeze-thaw experiment: The PCR reaction solution was frozen in a freezer at -20℃±5℃ for 1 hour, then removed and placed in a room temperature water bath to thaw completely (after thawing, it was gently mixed to ensure homogeneity of components). This constitutes one freeze-thaw cycle. After repeating this freeze-thaw cycle 4 times, the performance of the freeze-thawed PCR reaction solution was tested according to the method in Experiment (I) (referred to as "after freeze-thaw"), and the performance of the freshly prepared PCR reaction solution (referred to as "before freeze-thaw") was also tested. The CT values ​​are shown in Table 10.

[0105] Table 10

[0106] As can be seen from the results in Table 8-10, the performance of the PCR reaction solution provided by this invention is almost not reduced regardless of whether it is accelerated treatment at high temperature, storage under freezing conditions (-20±5℃) or freeze-thaw treatment.

[0107] (vi) The kit was prepared using the method described in Example 1, except that the primers and probes for viral hemorrhagic septicemia virus were replaced with those in Table 11 (the fluorescent reporter group and quencher group were the same as in Table 2), resulting in Kit B. Using Kit B, the simulated test samples were tested according to the method described in Experiment (i). The test results are as follows: Figure 3 As shown.

[0108] Table 11

[0109] contrast Figure 1 and Figure 3 The test results show that when using kit B, the fluorescence signal increment of viral hemorrhagic septicemia virus decreased slightly and the Ct value showed a slight delay, but the detection results still showed a typical S-type amplification curve and the Ct value was ≤38, which indicates that the primer and probe sequences selected by kit B have acceptable detection performance.

[0110] Comparative Example 1 The kit was prepared using the method described in Example 1, except that the target sequence of the infectious hematopoietic necrosis virus was replaced with the sequence shown in SEQ ID NO:29, and the corresponding primers and probes were replaced with those in Table 12 (the fluorescent reporter group and quencher group are the same as in Table 2), resulting in a comparative kit.

[0111]

[0112] Table 12

[0113] Using the method described in Experiment (I) of Example 2, the simulated test sample was tested using a comparison kit. The results are shown below. Figure 4 .contrast Figure 1 and Figure 4 The test results show that Figure 4 The fluorescence increment of infectious hematopoietic necrosis virus (IHHV) decreased and the Ct value was delayed. In addition, the IHHV primer probe had a significant impact on the amplification efficiency of viral hemorrhagic septicemia virus and Aeromonas salmonidae primer probe in the same tube. The fluorescence increment of the two targets decreased and the Ct value was delayed, indicating that selecting specific target sequences among the pathogens detected by each target can achieve better joint detection results.

[0114] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A composition for detecting pathogenic microorganisms in fish, characterized in that, The composition comprises at least one of the following primer pairs: Primer pair A: includes an upstream primer and a downstream primer targeting the target sequence of infectious hematopoietic necrosis virus, wherein the target sequence of infectious hematopoietic necrosis virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:

22. Primer pair B: includes an upstream primer and a downstream primer targeting the target sequence of infectious pancreatic necrosis virus, wherein the target sequence of infectious pancreatic necrosis virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:

23. Primer pair C: includes an upstream primer and a downstream primer targeting the target sequence of viral hemorrhagic septicemia virus, wherein the target sequence of viral hemorrhagic septicemia virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:

24. Primer pair D: includes an upstream primer and a downstream primer for the target sequence of Flavobacterium psychrophilum, wherein the target sequence of Flavobacterium psychrophilum has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:25; Primer pair E: includes an upstream primer and a downstream primer targeting the target sequence of Aeromonas salmonicida, wherein the target sequence of Aeromonas salmonicida has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:26; Primer pair F: includes an upstream primer and a downstream primer for the target sequence of Aeromonas hydrophila, wherein the target sequence of Aeromonas hydrophila has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:

27.

2. The composition according to claim 1, wherein, The nucleotide sequence of the target sequence of infectious hematopoietic necrosis virus is shown in SEQ ID NO:22; Preferably, primer pair A comprises primers with nucleotide sequences as shown in SEQ ID NO:1-2; And / or, the nucleotide sequence of the target sequence of infectious pancreatic necrosis virus is shown in SEQ ID NO:23; Preferably, primer pair B comprises primers with nucleotide sequences as shown in SEQ ID NO:4-5; And / or, the nucleotide sequence of the target sequence of viral hemorrhagic septicemia virus is shown in SEQ ID NO:24; Preferably, primer pair C comprises primers with nucleotide sequences as shown in SEQ ID NO:7-8 or SEQ ID NO:33-34; And / or, the nucleotide sequence of the target sequence of Flavobacterium psychrophilum is shown in SEQ ID NO:25; Preferably, primer pair D comprises primers with nucleotide sequences as shown in SEQ ID NO:10-11; And / or, the nucleotide sequence of the target sequence of Aeromonas salmonicida is shown in SEQ ID NO:26; Preferably, primer pair E comprises primers with nucleotide sequences as shown in SEQ ID NO:13-14; And / or, the nucleotide sequence of the target sequence of Aeromonas hydrophila is shown in SEQ ID NO:27; Preferably, primer pair F comprises primers with nucleotide sequences as shown in SEQ ID NO:16-17.

3. The composition according to claim 1 or 2, wherein, The composition may also include a quality control internal standard and a primer pair G for detecting the quality control internal standard, wherein the quality control internal standard is preferably selected from plant housekeeping genes; Preferably, the quality control internal standard is the Arabidopsis housekeeping gene, and preferably the target sequence of the quality control internal standard has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:28; Preferably, primer pair G comprises primers with nucleotide sequences as shown in SEQ ID NO:19-20.

4. The composition according to any one of claims 1-3, wherein, The composition further includes a nucleic acid probe, the nucleic acid probe comprising at least one of the following: Nucleic acid probe A includes probes targeting the target sequence of infectious hematopoietic necrosis virus; Nucleic acid probe B includes probes targeting the sequence of infectious pancreatic necrosis virus; Nucleic acid probe C includes probes targeting the target sequence of viral hemorrhagic septicemia virus; Nucleic acid probe D includes probes targeting the target sequence of Flavobacterium psychrophilum; Nucleic acid probe E includes probes targeting the target sequence of Aeromonas salmonii; Nucleic acid probe F includes probes targeting the sequence of Aeromonas hydrophila. Nucleic acid probe G includes probes targeting the target sequence of the quality control internal standard; Preferably, nucleic acid probe A comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:3; Preferably, nucleic acid probe B comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:6; Preferably, nucleic acid probe C comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:9 or SEQ ID NO:35; Preferably, nucleic acid probe D comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:12; Preferably, nucleic acid probe E comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:15; Preferably, the nucleic acid probe F comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:18; Preferably, the nucleic acid probe G comprises a nucleic acid probe with a nucleotide sequence as shown in SEQ ID NO:

21.

5. The composition according to claim 4, wherein, The nucleic acid probe further includes a reporter group modified on the nucleic acid probe, preferably a fluorescent reporter group; Preferably, the nucleic acid probe further includes a fluorescence quenching group modified on the nucleic acid probe.

6. A reagent kit, characterized in that, The kit comprises the composition according to any one of claims 1-5.

7. The kit according to claim 6, wherein, The kit also contains at least one of the following: buffer, enzyme, ribonuclease inhibitor, magnesium source, and deoxyribonucleotide triphosphate; Preferably, the buffer solution comprises tris(hydroxymethyl)aminomethane hydrochloride buffer; Preferably, the enzyme includes at least one of reverse transcriptase, UNG enzyme, and DNA polymerase; More preferably, the kit also includes a positive control and a negative control.

8. The use of the composition of any one of claims 1-5, or the kit of claim 6 or 7, in the detection of fish pathogens in samples, particularly in the detection of fish pathogens in salmonid samples.

9. A method for detecting pathogenic microorganisms in fish samples, characterized in that, The method includes amplifying nucleic acid in the sample to be tested using the composition of any one of claims 1-5 or the kit of claim 6 or 7, wherein the fish pathogenic microorganism is selected from at least one of infectious hematopoietic organ necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, Flavobacterium psychrophilum, Aeromonas salmonidae, and Aeromonas hydrophila.

10. The method according to claim 9, wherein, The method includes: (1) Extract nucleic acid from the sample to be tested; (2) Amplify the nucleic acid extracted in step (1).

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