A kit and method for rapid detection of vibrio columnar, vibrio mediterranei and vibrio harveyi

Abstract

The application provides a kit and method for rapidly detecting Vibrio tapetis, Vibrio mediterranei and Vibrio harveyi. First, a series of primer probe combinations are designed for the three pathogenic bacteria, and then through multiple rounds of screening, the triple amplification primer probe group with the highest detection sensitivity is found. Meanwhile, the amplification system is optimized, and by adding oligopeptide and DMSO in the A Buffer, the amplification efficiency is significantly improved, the detection sensitivity and specificity are improved, the minimum detectable sample is 0.5 copies / μL, the specificity is good, and the detection time only needs 10-20 minutes. The rapid detection of Vibrio tapetis, Vibrio mediterranei and Vibrio harveyi has great significance for cutting off the transmission route and reducing the risk of factory breeding of Hemifusus ruber.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to a kit and method for rapid detection of Vibrio tarda, Vibrio thalassemia, and Vibrio harveyi. Background Technology

[0002] Vibrio infection is currently the most prevalent disease affecting the farming of Brachiosa serrata in Hainan Province. Every year from April to May and October to November, farmed Brachiosa serrata are susceptible to "acute mortality syndrome" caused by Vibrio talus, Vibrio thalassemia, or Vibrio harveyi, leading to large-scale mortality and a reduction in annual yield of over 40%. To enable timely and effective diagnosis of the disease and prompt intervention, there is an urgent need to establish a highly specific, highly sensitive, time-efficient, and simple method for the rapid detection of these three pathogens simultaneously.

[0003] Recombinase-aided amplification (RAA) is a rapid isothermal nucleic acid amplification technique. Recombinases obtained from bacteria or fungi bind tightly to primer DNA at room temperature, forming a recombinase / primer complex. This complex invades the double-stranded DNA template, opening the double strands at the invasion site. Simultaneously, single-strand binding proteins bind to the opened single strands, maintaining the template in an open state. The recombinase / primer complex scans the double strands. When the primer finds a perfectly matching complementary sequence on the template, the complex disintegrates, and DNA polymerase binds to the 3' end of the primer, initiating the synthesis of a new strand. This new strand can then serve as a template, and the amplification product grows exponentially, completing the amplification of the target gene. Fluorescently labeled probes bind to the amplification products. When the probe is cleaved by exonuclease, it emits a fluorescent signal, allowing for real-time monitoring of the amplification process. Therefore, multiplex RAA amplification can be considered for the rapid detection of three pathogens: Vibrio talar, Vibrio thalassemia, and Vibrio harveyi.

[0004] However, multiplex RAA is highly dependent on the target sequence of the amplified nucleic acid, primer design, and amplification system formulation. Therefore, it is necessary to find the most suitable primers and amplification system design to achieve high specificity and high sensitivity detection of three pathogens simultaneously: Vibrio tatarsi, Vibrio thalassemia, and Vibrio harveyi. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a rapid detection kit and method for Vibrio talar, Vibrio thalassemia, and Vibrio harveyi. Firstly, hundreds of primer-probe combinations were designed for these three pathogens. After multiple rounds of screening, the triple amplification primer-probe set with the highest detection sensitivity was selected. Simultaneously, the amplification system was optimized by adding oligopeptides and DMSO to Buffer A, significantly improving amplification efficiency and further enhancing detection sensitivity and specificity. The minimum detectable sample size is 0.5 copies / μL, exhibiting good specificity and requiring only 10–20 minutes for detection. Rapid detection of Vibrio talar, Vibrio thalassemia, and Vibrio harveyi is of great significance in cutting off transmission routes and reducing the risks associated with factory-farmed Brachydius sinensis.

[0006] The sensitivity of PCR multiplex amplification is almost identical to that of singlex amplification. However, RAA singlex amplification has high sensitivity, doublex amplification decreases sensitivity, and triplex amplification decreases sensitivity sharply. Therefore, to perform RAA triplex amplification while maintaining high detection sensitivity, it is necessary not only to select a suitable primer-probe set but also to modify the RAA amplification system. This invention significantly improves the detection sensitivity of RAA triplex amplification by selecting the optimal primer-probe set and improving the composition of A Buffer, while maintaining very good specificity.

[0007] On one hand, the present invention provides a primer-probe combination for triple amplification detection of Vibrio pyrrhizosus, Vibrio thaliana, and Vibrio harveyi, comprising the following upstream primer, downstream primer, and probe:

[0008] This invention selects highly specific fragments from the *Vibrio thaliana* pyrH gene, *Vibrio tarda* gyrB gene, and *Vibrio harveyi* hemolysin gene as target amplification regions. The nucleotide sequences of the amplification region of the *Vibrio thaliana* pyrH gene are shown in Seq ID NO. 10, the *Vibrio tarda* gyrB gene amplification region in Seq ID NO. 11, and the *Vibrio harveyi* hemolysin gene amplification region in Seq ID NO. 12. A series of primer and probe sequences were designed for each of these three target amplification regions, resulting in hundreds of primer and probe combinations. After multiple rounds of screening, the triple amplification primer and probe set with the highest detection sensitivity, as shown in the table above, was selected.

[0009] Furthermore, the probes for Vibrio tatarsus, Vibrio thaliana, and Vibrio harveyi are labeled with different fluorescent groups.

[0010] In some embodiments, the probes for Vibrio tatarica are labeled with the FAM fluorescent group; the probes for Vibrio thaliana are labeled with the ROX fluorescent group; and the probes for Vibrio harveyi are labeled with the CY5 fluorescent group.

[0011] The specific primer and probe sequences are shown in the table below:

[0012] On the other hand, the present invention provides a kit for triple amplification detection of Vibrio tarda, Vibrio thaliana and Vibrio harveyi, comprising the primer and probe combination as described above.

[0013] Furthermore, it also includes A Buffer; the A Buffer contains oligopeptides, the amino acid sequence of which is any one or more selected from Seq ID NO.13 to Seq ID NO.17.

[0014] Oligopeptides are polypeptides composed of fewer than 20 amino acids. This invention demonstrates that adding oligopeptides to the RAA amplification system can significantly improve detection sensitivity. This is likely because oligopeptides help maintain system stability, thereby enhancing detection sensitivity.

[0015] In some methods, oligopeptides can be identified as oligopeptide-1 (Seq ID NO.13), oligopeptide-2 (Seq ID NO.14), oligopeptide-3 (Seq ID NO.15), oligopeptide-4 (Seq ID NO.16), and oligopeptide-5 (Seq ID NO.17).

[0016] Furthermore, the amino acid sequence of the oligopeptide is Seq ID NO.13.

[0017] Different oligopeptides have different effects on improving detection sensitivity, and the most preferred oligopeptides have an amino acid sequence as shown in Seq ID NO.13.

[0018] Furthermore, the A Buffer also contains DMSO (dimethyl sulfoxide).

[0019] DMSO promotes the dissolution of oligopeptides. Therefore, adding DMSO to a buffer containing oligopeptides can further improve detection sensitivity.

[0020] Furthermore, the A Buffer contains polyethylene glycol, Tris-HCl, DMSO, and oligopeptides; the kit also includes a B Buffer containing magnesium acetate, magnesium chloride, and manganese chloride.

[0021] Furthermore, the kit also includes RAA reaction powder, positive control, and negative control; the amplification conditions for RAA are: 39–42°C for 10–20 min.

[0022] In some methods, the components of the RAA reaction powder include recombinase, single-stranded binding protein, DNA polymerase, exonuclease, ATP, dNTPs, creatine kinase, disodium creatine phosphate, trehalose, mannitol, etc.

[0023] In some embodiments, the positive control is a positive plasmid containing the target amplification region sequence of Vibrio tatarsus, Vibrio thaliana, and Vibrio harveyi, wherein the positive plasmid contains the nucleotide sequences shown in Seq ID NO.10, Seq ID NO.11, and Seq ID NO.12.

[0024] In some methods, the negative control is purified water.

[0025] In another aspect, the present invention provides a method for triple amplification detection of Vibrio tarda, Vibrio thalassemia, and Vibrio harveyi, wherein the method uses the primer and probe combination as described above, or uses the kit as described above to perform RAA amplification on the sample and then detect it.

[0026] In some embodiments, the method includes the following steps: ① using the nucleic acid of the sample to be tested as a template; ② performing a RAA reaction using the kit described in this invention; ③ analyzing the sample to be tested based on the real-time fluorescence amplification results to determine whether Vibrio tatarsi, Vibrio thalassemia, and Vibrio harveyi are present in the sample to be tested.

[0027] The specific steps are as follows: ① Extract nucleic acid from the sample to be tested using a nucleic acid extraction kit, or treat the sample with a sample release agent, or release the nucleic acid from the sample by boiling; ② Use the nucleic acid of the sample to be tested as a template, and perform a RAA reaction in the presence of RAA reaction powder (containing primers and probes for detecting Vibrio talus, Vibrio thalassemia, and Vibrio harveyi), Buffer A, and Buffer B; ③ Analyze the real-time fluorescence amplification results. Applicable instruments and equipment include various models of fluorescence PCR instruments, real-time fluorescence thermostats, etc. The nucleotide sequences of the primer-probe combination are shown in SEQ ID NO.1 to SEQ ID NO.9. Preferably, the RAA reaction conditions are: 39–42℃, time 10–20 min.

[0028] In another aspect, the present invention provides the use of oligopeptides in the preparation of RAA amplification reagents for triple amplification detection of Vibrio tarda, Vibrio thaliana and Vibrio harveyi, wherein the amino acid sequence of the oligopeptide is any one or more selected from Seq ID NO.13 to Seq ID NO.17.

[0029] The kit and detection method provided by this invention can quickly, conveniently, efficiently and specifically detect whether a sample contains Vibrio tarda, Vibrio thaliana, and Vibrio harveyi. It is suitable for clinical identification and detection, animal disease detection, and can also monitor the presence of Vibrio tarda, Vibrio thaliana, and Vibrio harveyi in the environment (such as animal excrement sites).

[0030] The beneficial effects of this invention are as follows: (1) Select the fragments with high specificity and low mutual interference in Vibrio thaliana pyrH gene, Vibrio tarda gyrB gene and Vibrio harveyi hemolysin gene as the target amplification region, design a series of primer probe sequences, obtain hundreds of primer probe combinations, and then find the triple amplification primer probe set with the highest detection sensitivity through multiple rounds of screening. (2) Adding oligopeptides to the A Buffer of the RAA amplification system can help maintain the stability of the system, thereby improving the detection sensitivity. At the same time, the sequences of oligopeptides were screened and the optimal oligopeptide sequence was found. (3) Adding DMSO to A Buffer, which contains oligopeptides, helps to further improve the detection sensitivity; (4) It is time-saving, convenient and fast, with a short detection cycle, requiring only 10 to 20 minutes per round of detection; it can simultaneously use 3 fluorescence channels to detect Vibrio tarda, Vibrio thalassemia and Vibrio harveyi, with high detection efficiency; (5) It can detect samples as low as 0.5 copies / μL, and also has very good specificity; (6) Wide applicability and simple identification: Applicable instruments and equipment include various models of fluorescence PCR instruments, real-time fluorescence thermostats, etc. The appearance of a specific amplification curve indicates a positive result; (7) Rapid detection of Vibrio tatarsus, Vibrio thaliana and Vibrio harveyi is of great significance in cutting off the transmission route and reducing the risk of factory farming of the spotted snail. Detailed Implementation

[0031] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and are not intended to limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0032] Example 1: Validation of the rapid detection kit, detection method, and detection results of Vibrio tarda, Vibrio thalassemia, and Vibrio harveyi provided by the present invention. 1. This embodiment provides a rapid detection kit for Vibrio tarda, Vibrio thalassemia, and Vibrio harveyi. The kit includes a primer and probe set, RAA reaction powder, A Buffer, B Buffer, positive control, and negative control.

[0033] Primer and probe set: Vibrio truncatum primer and probe (0.13 μL, 50 mM), Vibrio thaliana primer and probe (0.13 μL, 50 mM), Vibrio harveyi primer and probe (0.13 μL, 50 mM), primer and probe sequences are shown in Table 1; Table 1. Primer and probe sequence

[0034] RAA Reaction Powder: Recombinase (6.5 μg / tube), Single-stranded binding protein (40 μg / tube), DNA polymerase (3.5 μg / tube), Exonuclease (5.0 μg / tube), ATP (0.075 mg / tube), dNTPs (13.2 ng / tube), Creatine kinase (5.0 μg / tube), Creatine phosphate disodium salt (0.65 mg / tube), Trehalose (1.3 mg / tube), Mannitol (1.25 mg / tube); The components of Buffer A are: polyethylene glycol (PEG) (0.2 mg / μL), Tris-HCl (6.05 μg / μL), DMSO (15 μg / μL), and oligopeptide (SEQ ID NO.13) (1.5 μg / μL). B Buffer consists of: magnesium acetate tetrahydrate (MgAc2) (60 μg / μL), magnesium chloride (MgCl2) (2.66 μg / μL), and manganese chloride (MnCl2) (3.78 μg / μL). The positive control provided in this embodiment is a positive plasmid containing the target amplification region sequence of Vibrio tatarsus, Vibrio thaliana, and Vibrio harveyi (the positive plasmid contains the nucleotide sequences shown in Seq ID NO.10, Seq ID NO.11, and Seq ID NO.12), and the negative control is purified water.

[0035] 2. The kit of this embodiment is used to test the sample. The specific steps are as follows: 2.1 Extraction of nucleic acid from samples Method 1: Genomic DNA was extracted from the sample using a tissue genomic DNA extraction kit, following the instructions.

[0036] Method 2: Samples are treated with a nucleic acid release agent without extracting nucleic acid; the lysate is used directly as a template.

[0037] 2.2 Preparation of RAA reaction system: Each test sample corresponds to one RAA reaction dry powder tube. The reaction components and the volume added in each RAA reaction tube are shown in Table 2.

[0038] Table 2. Preparation of RAA Reaction System

[0039] To prevent the reagents used from becoming ineffective or contaminated, positive and negative controls were set up (the positive control is a plasmid containing three amplification regions, and the negative control is water, which is added to the system as a template; the dosage is shown in Table 2). The design of the negative control can effectively verify whether the reagents used are contaminated and avoid false positives. The design of the positive control can effectively verify the effectiveness of the reagents used and avoid false negatives.

[0040] 2.3 Invert the prepared RAA reaction tube 6-8 times to thoroughly mix the reaction solution, and centrifuge at low speed for a few seconds to ensure all the reaction solution reaches the bottom of the tube. Place the reaction tube in a fluorescence PCR instrument or a constant temperature fluorescence instrument, set the reaction temperature to 39℃, and the reaction time to 20 min. Determine the result based on whether a specific amplification curve appears.

[0041] 3. Verification and analysis of test results: 3.1 Accuracy Verification To verify that the primers, probes, and reagents described in this embodiment can be used normally under actual conditions, the kit provided in this embodiment was used to test actual samples. The Hainan Academy of Marine and Fisheries Sciences provided 25 aquatic samples that had already been confirmed by PCR testing. The reaction temperature was set at 42℃, and the reaction time was 10 min. The test results are shown in Table 3. The test results of the kit provided in this embodiment are consistent with the positive and negative results of the samples, with a concordance rate of 100%.

[0042] Table 3. Results of actual sample testing using the kit provided in this embodiment.

[0043] 3.2 Sensitivity Test Plasmids containing amplified regions of Vibrio tatarsus, Vibrio thaliana, and Vibrio harveyi were cultured and extracted. The concentration of positive plasmids was measured using NanoDrop, and sensitivity tests were performed by diluting the plasmids to six concentration gradients: 100 copies / μL, 10 copies / μL, 1 copy / μL, 0.5 copies / μL, 0.3 copies / μL, and 0.1 copies / μL. Twenty replicates were performed at each concentration. The results are shown in Table 4. Table 4. Sensitivity test results of the reagent kit provided in this embodiment.

[0044] The test results show that the triple fluorescence detection kit provided in this embodiment has a detection sensitivity of up to 0.5 copies / μL, or 500 copies / mL, indicating that the detection kit and detection method provided in this embodiment have high sensitivity for the differential diagnosis of Vibrio tatarsus, Vibrio thalassemia, and Vibrio harveyi.

[0045] 3.3 Specificity test To test the specificity of the kit provided in this embodiment, other bacterial strain samples were tested. The test results are shown in Table 5. The test results of the specificity test samples were all negative, indicating that the method and kit provided in this embodiment have high specificity.

[0046] Table 5. Specificity test results of the reagent kit provided in this embodiment

[0047] As can be seen from Table 5, the kit provided in this embodiment is compatible with Staphylococcus aureus (Staphylococcus aureus). Staphylococcus golden Staphylococcus epidermidis ( Staphylococcus epidermidis Helicobacter pylori ( Helicobacter pylori ), Escherichia coli ( Escherichia coli ), Shigella dysenteriae ( Shigella dysenteriae ), Mermaid luminescent bacteria ( Photobacterium damselae ), Vibrio cholerae ( Cholera vibrio Vibrio parahaemolyticus ( Parahemolytic Vibrio ), Rotiferous Vibrio ( Vibrio rotifera Vibrio croakerii ( ) Vibrio Pontic ), Vibrio alginolyticus ( Vibrio alginolyticus Alternaria mellea ( Alteromonas Macleod's ), Indian microbacterium ( Exiguobacterium indicum ), Vibrio lobsterii ( Vibrio panuliri ), Vibrio xuei ( Vibrio xvii ), Vibrio Owens ( Vibrio owensii There is no cross-reactivity with any of them, and they have very good specificity.

[0048] Example 2: Screening of primer and probe sets This embodiment references the published gene sequences of *Vibrio thaliana* pyrH, *Vibrio tarda* gyrB, and *Vibrio harveyi* hemolysin in GenBank, and uses DNAMAN 6.0 software for multiple sequence alignment. Fragments with high specificity for the three vibrio species and low likelihood of mutual interference were selected as the target amplification regions. The nucleotide sequence of the amplified region of the *Vibrio thaliana* pyrH gene is shown in Seq ID NO. 10, the nucleotide sequence of the amplified region of the *Vibrio tarda* gyrB gene is shown in Seq ID NO. 11, and the nucleotide sequence of the *Vibrio harveyi* hemolysin gene is shown in Seq ID NO. 12.

[0049] Numerous experiments have shown that different primers have a certain impact on the efficiency and sensitivity of RAA fluorescence amplification. Therefore, in this embodiment, the following primers were initially designed for screening for each of the three target gene fragments mentioned above.

[0050] 1. Primers and probes for Vibrio thaliana: The Vibrio thaliana probe VM-Probe is labeled with ROX fluorescence and has four modification sites: ROX-dT, BHQ1-dT, THF, and C3 Spacer. The specific sequence is as follows: GTGTTTGCGACGATTACAACTGGGC[ROX-dT][THF]A[BHQ1-dT]GCTATTCGTGAATTAC-C3Spacer Ten upstream primers and ten downstream primers for Vibrio thaliana were designed and screened, as shown in Table 6. Table 6. Upstream and downstream primer design for Vibrio thaliana

[0051] 2. Primers and probes for Vibrio tali: The tower-shaped Vibrio probe gyrB-Probe is labeled with FAM fluorescence and has four modification sites: FAM-dT, BHQ1-dT, THF, and C3 Spacer. The specific sequence is as follows: TTCAGCAGTACAACGCGGGTATTAAGC[FAM-dT][THF]G[BHQ1-dT]GGATCGCATGAG- C3Spacer Eleven upstream primers and nine downstream primers were designed and screened for Vibrio talus, as shown in Table 7. Table 7. Upstream and downstream primer design for Vibrio pyrrhizosus

[0052] 3. Primers and probes for Vibrio harveyi: The Vibrio harveyi probe VHV-Probe is labeled with FAM fluorescence and has four modification sites: CY5-dT, BHQ2-dT, THF, and C3 Spacer. The specific sequence is as follows: GCGGATCTTGTTGATTTCTTCT[Cy5-dt]G[THF]G[BHQ2-dT]CGAGTAGGTAAAC- C3Spacer Eight upstream primers and seven downstream primers were designed and screened for Vibrio harveyi, as shown in Table 8. Table 8. Upstream and downstream primer design for Vibrio harveyi

[0053] First round of primer screening: Prepare the reaction system according to Table 9 for the first round of primer screening.

[0054] Table 9. Preparation of RAA Reaction System

[0055] Invert the prepared RAA reaction tube 6-8 times to thoroughly mix the reaction solution, and centrifuge at low speed for a few seconds to ensure all the reaction solution reaches the bottom of the tube. Place the reaction tube in a fluorescence PCR instrument, set the reaction temperature to 39℃, and the reaction time to 20 minutes.

[0056] First-round primer screening method: For each of the three Vibrio species, upstream and downstream primers were paired and combined, and the two most sensitive primer combinations were selected from each species. For Vibrio thaliana, there are 10 upstream primers (VM-F1 to VM-F10) and 10 downstream primers (VM-R1 to VM-R10), with a total of 100 combinations. The combinations with the highest sensitivity are VM-F5 + VM-R9 and VM-F10 + VM-R10. Eleven upstream primers (gyrB-F1 to gyrB-F11) and nine downstream primers (gyrB-R1 to gyrB-R9) were used for Vibrio tali, resulting in a total of 99 combinations. The combinations with the highest sensitivity were gyrB-F5 + gyrB-R1 and gyrB-F10 + gyrB-R9. For Vibrio harveyi, there are 8 upstream primers (VHV-F1 to VHV-F8) and 7 downstream primers (VHV-R1 to VHV-R7), resulting in a total of 56 combinations. The combinations with the highest sensitivity are VHV-F3 + VHV-R1 and VHV-F6 + VHV-R6. Second round of primer screening: Two sets of primers with the highest sensitivity for each of the three Vibrio species obtained in the first round of screening (a total of 8 combinations) were used to prepare reaction systems according to Table 10 for the second round of primer screening.

[0057] Table 10. Preparation of RAA Reaction System

[0058] Invert the prepared RAA reaction tube 6-8 times to thoroughly mix the reaction solution, and centrifuge at low speed for a few seconds to ensure all the reaction solution reaches the bottom of the tube. Place the reaction tube in a fluorescence PCR instrument, set the reaction temperature to 39℃, and the reaction time to 20 minutes.

[0059] The specific screening process and results of the second round of primers are shown in Table 11.

[0060] Table 11. Specific primer combinations and sensitivity detection results for the second round.

[0061] The seventh primer combination was selected as the best, specifically: the primer combination of Vibrio thaliana VM-F10+VM-R10, Vibrio tauridii gyrB-F10+gyrB-R9, and Vibrio harveyi VHV-F3+VHV-R1, with a sensitivity of up to 500 copies / mL.

[0062] Example 3: The effect of oligopeptides on detection results This embodiment uses the method provided in Example 1 to perform triple amplification detection of Vibrio tarda, Vibrio thalassemia, and Vibrio harveyi. The RAA buffer is categorized into seven cases based on whether it contains oligopeptides: 1. No oligopeptides; 2. Contains oligopeptide one (Seq ID NO. 13); 3. Contains oligopeptide two (Seq ID NO. 14); 4. Contains oligopeptide three (Seq ID NO. 15); 5. Contains oligopeptide four (Seq ID NO. 16); 6. Contains oligopeptide five (Seq ID NO. 17); 7. No oligopeptides, contains bovine serum albumin (BSA). Details are shown in Table 12. All seven cases contain DMSO in the A buffer. After RAA amplification using different A buffers, the content of the three bacteria was detected, and the detection sensitivity results are shown in Table 12.

[0063] Table 12. Effect of oligopeptides in Buffer A on detection sensitivity

[0064] As shown in Table 12, when A Buffer contains oligopeptides (cases 2-6), the detection sensitivity can be significantly improved compared to case 1. Case 2 (containing oligopeptide Seq ID NO.13) is the most preferred, with the highest detection sensitivity. The detection sensitivity for all three bacteria can reach 0.5 copies / μL.

[0065] Example 4: The impact of DMSO on detection results This embodiment uses the method provided in Example 1 to perform triple amplification detection of Vibrio tarda, Vibrio thalassemia, and Vibrio harveyi. The RAA buffer contains oligopeptide Seq ID NO.13, and the samples are divided into two groups based on whether or not DMSO is present. The effect of DMSO on the detection results is investigated. The results are shown in Table 13.

[0066] Table 13. Effect of DMSO on detection sensitivity

[0067] As shown in Table 13, the presence of DMSO in Buffer A can significantly improve the detection sensitivity. This may be because DMSO can improve the solubility of oligopeptide Seq ID NO.13, thereby improving the stability of the RAA system and increasing the detection sensitivity.

[0068] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer-probe combination for RAA triple amplification detection of Vibrio pyrrhizosum, Vibrio thalassemia, and Vibrio harveyi, characterized in that, This includes the following upstream primers, downstream primers, and probes: ； The probe labeling for Vibrio pyrophorus contains FAM fluorescence and has four modification sites: FAM-dT, BHQ1-dT, THF, and C3 Spacer, with the following specific sequences: TTCAGCAGTACAACGCGGGTATTAAGC[FAM-dT][THF]G[BHQ1-dT]GGATCGCATGAG- C3Spacer; The probe for Vibrio thaliana is labeled with ROX fluorescence, with a total of four modification sites: ROX-dT, BHQ1-dT, THF, and C3Spacer, and the specific sequences are as follows: GTGTTTGCGACGATTACAACTGGGC[ROX-dT][THF]A[BHQ1-dT]GCTATTCGTGAATTAC-C3Spacer; The probe labeling Cy5 fluorescent strain of Vibrio harveyi has four modification sites: CY5-dT, BHQ2-dT, THF, and C3Spacer, with the following specific sequences: GCGGATCTTGTTGATTTCTTCT[Cy5-dt]G[THF]G[BHQ2-dT]CGAGTAGGTAAAC- C3 Spacer.

2. A kit for RAA triple amplification detection of Vibrio pyrrhosa, Vibrio thalassemia, and Vibrio harveyi, characterized in that, Includes the primer-probe combination as described in claim 1.

3. The kit according to claim 2, characterized in that, It also includes A Buffer; the A Buffer contains oligopeptides, the amino acid sequences of which are any one or more selected from Seq ID NO.13 to Seq ID NO.

17.

4. The kit according to claim 3, characterized in that, The amino acid sequence of the oligopeptide is Seq ID NO.

13.

5. The kit according to claim 4, characterized in that, The A Buffer also contains DMSO.

6. The reagent kit as described in claim 5, characterized in that, The A Buffer contains polyethylene glycol, Tris-HCl, DMSO, and oligopeptides; it also includes a B Buffer containing magnesium acetate, magnesium chloride, and manganese chloride.

7. The kit according to claim 6, characterized in that, It also includes RAA reaction powder, positive control and negative control; the amplification conditions for RAA are: 39-42℃, time 10-20min.

8. The use of oligopeptides in the preparation of RAA amplification reagents for triple amplification detection of Vibrio pyrrhizosum, Vibrio thaliana, and Vibrio harveyi, characterized in that, The amino acid sequence of the oligopeptide is selected from any one or more of Seq ID NO.13 to Seq ID NO.17, and the RAA amplification reagent includes the primer-probe combination as described in claim 1.

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