Marker and method for detecting five pathogenic vibrios of aquatic animals and application

Abstract

The invention discloses a marker and a method for detecting five pathogenic vibrios of aquatic animals and application, and belongs to the technical field of pathogenic vibrio detection. The coding gene of the difunctional diguanylate cyclase / phosphodiesterase is found for the first time and has specificity in five aquatic animal pathogenic vibrios, and rapid detection of the five aquatic animal pathogenic vibrios can be achieved through specific primers designed for the gene; except vibrio harveyi, the other four pathogenic vibrios all have two pairs of specific primers, so that the purpose of dual verification can be achieved during detection, and false negative caused by gene deletion can be effectively avoided; the provided method is high in specificity; the detection time is short; the operation is simple and easy; the detection cost is low; the lowest detection limit is low, and the lowest detection limit range of the nine pairs of specific primers is 0.0157 ng / [mu] L-6. 1813 ng / [mu] L; the method is suitable for large-scale daily detection of basic farms.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic vibrio detection, and in particular to a biomarker, method and application for detecting pathogenic vibrios of five aquatic animals. Background Art

[0002] Vibrio belongs to the family Vibrionaceae and order Vibrionales taxonomically, and is a Gram-negative short bacillus. The cell morphology is mostly curved or straight rod-shaped, and the single polar flagellum endows it with good motility. Vibrio microorganisms have thermophilic characteristics and are facultative anaerobes. They are widely distributed in freshwater, estuarine and marine ecosystems, especially the marine environment as the dominant habitat, and often adhere to the surfaces of marine animals and plants in the form of biofilms. As opportunistic pathogens, vibrios cause infections in aquatic animals through ways such as adhesion and colonization, host ingestion and filter-feeding behavior. When the environmental factors of the aquaculture water body (such as temperature, salinity, organic matter content) are suitable, their populations can grow exponentially, leading to the outbreak of vibriosis in aquatic animals, and in severe cases, it can cause large-scale deaths of the aquaculture population, causing significant economic losses to the aquaculture industry.

[0003] Among the pathogenic vibrios of aquatic animals, Vibrio harveyi, Vibrio cholerae, Vibrio vulnificus, Vibrio anguillarum and Vibrio parahaemolyticus are the main pathogens. It should be noted that Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus have the characteristics of zoonosis, which can not only cause diseases in aquatic animals, but also be the main pathogenic species of human vibrio infections. Vibrio anguillarum and Vibrio harveyi are typical pathogens of vibriosis in aquaculture. Given the characteristics of vibrios being ubiquitous in the environment, highly transmissible and causing great pathogenic harm, establishing an effective daily monitoring system is crucial for the prevention and control of aquaculture diseases.

[0004] Current vibrio detection still mainly relies on traditional microbiological methods, and its standard process includes enrichment culture on selective medium, colony isolation and purification, Gram staining microscopy and a series of biochemical identifications. However, this method has significant limitations: the operation process is cumbersome, the labor intensity is high, the detection sensitivity is poor, and the identification between species within the genus and between genera outside the genus is greatly affected by the operator, making it difficult to meet the rapid and large-scale daily detection needs of grass-roots farms. Due to the virulence genes and hemolysin genes of Vibrio strains, they are prone to dynamic evolution characteristics due to deletion, point mutation or horizontal gene transfer. PCR detection techniques targeting such genes are prone to false negative or false positive problems due to target loss or sequence variation, resulting in misdetection. In addition, some detection schemes have not established a strict cross-validation system, and there are still controversies about the specific amplification efficiency for other bacterial species. Therefore, developing new non-virulence-dependent molecular detection targets has important scientific value for improving the accuracy and applicability of vibrio species identification technology. Summary of the Invention

[0005] The object of the present invention is to provide a marker, method and application for detecting five pathogenic vibrios of aquatic animals, so as to solve the problems of the current detection method, such as cumbersome operation process, high labor intensity, poor sensitivity, easy occurrence of false negatives or false positives, and difficulty in meeting the rapid and large-scale daily detection needs of grass-roots farms.

[0006] To achieve the above object, the present invention provides a marker for detecting five pathogenic vibrios of aquatic animals, and the five pathogenic vibrios of aquatic animals are Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio anguillarum and Vibrio harveyi;

[0007] The marker is the coding gene of bifunctional diguanylate cyclase / phosphodiesterase, and the CDS sequences of the coding genes of the specific bifunctional diguanylate cyclase / phosphodiesterase of the 5 pathogenic vibrios are shown in SEQ ID NO.1 to SEQ ID NO.9 respectively.

[0008] Preferably, the specific primers for the coding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio parahaemolyticus are VP-1539-F, VP-1539-R and VP-2040-F, VP-2040-R; the amplification fragment length of the primer pair VP-1539-F and VP-1539-R is 699bp; the amplification fragment length of the primer pair VP-2040-F and VP-2040-R is 649bp; the sequence of VP-1539-F is shown in SEQ ID NO.10, the sequence of VP-1539-R is shown in SEQ ID NO.11, the sequence of VP-2040-F is shown in SEQ ID NO.12, and the sequence of VP-2040-R is shown in SEQ ID NO.13.

[0009] Preferably, the specific primers for the coding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio cholerae are VC-1833-F, VC-1833-R and VC-2055-F, VC-2055-R; the amplification fragment length of the primer pair VC-1833-F and VC-1833-R is 397bp; the amplification fragment length of the primer pair VC-2055-F and VC-2055-R is 208bp; the sequence of VC-1833-F is shown in SEQ ID NO.14, the sequence of VC-1833-R is shown in SEQ ID NO.15, the sequence of VC-2055-F is shown in SEQ ID NO.16, and the sequence of VC-2055-R is shown in SEQ ID NO.17.

[0010] Preferably, the specific primers for the coding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio vulnificus are VV-1599-F, VV-1599-R and VV-1776-F, VV-1776-R; the amplified fragment length of the primer pair VV-1599-F and VV-1599-R is 455 bp; the amplified fragment length of the primer pair VV-1776-F and VV-1776-R is 569 bp; the sequence of VV-1599-F is as shown in SEQ ID NO.18, the sequence of VV-1599-R is as shown in SEQ ID NO.19, the sequence of VV-1776-F is as shown in SEQ ID NO.20, and the sequence of VV-1776-R is as shown in SEQ ID NO.21.

[0011] Preferably, the specific primers for the coding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio anguillarum are VN-1491-F, VN-1491-R and VN-1833-F, VN-1833-R; the amplified fragment length of the primer pair VN-1491-F and VN-1491-R is 323 bp; the amplified fragment length of the primer pair VN-1833-F and VN-1833-R is 853 bp; the sequence of VN-1491-F is as shown in SEQ ID NO.22, the sequence of VN-1491-R is as shown in SEQ ID NO.23, the sequence of VN-1833-F is as shown in SEQ ID NO.24, and the sequence of VN-1833-R is as shown in SEQ ID NO.25.

[0012] Preferably, the specific primers for the coding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio harveyi are VH-1821-F, VH-1821-R; the amplified fragment length of the primer pair VH-1821-F and VH-1821-R is 521 bp; the sequence of VH-1821-F is as shown in SEQ ID NO.26, and the sequence of VH-1821-R is as shown in SEQ ID NO.27.

[0013] A method for detecting five kinds of pathogenic Vibrio in aquatic animals, using the above-mentioned markers for detecting five kinds of pathogenic Vibrio in aquatic animals, comprises the following steps:

[0014] Add the bacteria to be tested into a dry heater, heat at 100 °C for 10 min, use the obtained solution as a template for PCR to detect whether the coding gene of bifunctional diguanylate cyclase / phosphodiesterase exists. If the coding gene of bifunctional diguanylate cyclase / phosphodiesterase exists, it indicates that the test substance is one or several of the five kinds of pathogenic Vibrio in aquatic animals; if the coding gene of bifunctional diguanylate cyclase / phosphodiesterase does not exist, it indicates that the test substance does not contain the five kinds of pathogenic Vibrio in aquatic animals.

[0015] Preferably, the PCR system is as follows: 0.8 μL of template, 0.8 μL of each of the upstream and downstream primers, 10 μL of 2×Rapid Taq MasterMix, and sterile ultrapure water is added to make up to 20 μL;

[0016] The PCR amplification program is: 95°C for 3 min; 95°C for 15 s, 61 ± 0.5°C for 15 s, 72°C for 2 s, for 25 cycles; 72°C for 5 min; keep at 4°C.

[0017] Use of the marker for detecting five pathogenic Vibrio species in aquatic animals for detecting pathogenic Vibrio.

[0018] A kit for simultaneously detecting five pathogenic Vibrio species in aquatic animals, the kit includes the marker for detecting five pathogenic Vibrio species in aquatic animals; the five pathogenic Vibrio species in aquatic animals are Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio anguillarum, and Vibrio harveyi.

[0019] Therefore, a marker, method and application for detecting five pathogenic Vibrio species in aquatic animals provided by the present invention have the following specific technical effects:

[0020] (1) The present invention first discovers that the coding gene of bifunctional diguanylate cyclase / phosphodiesterase is specific among five pathogenic Vibrio species in aquatic animals. By using specific primers designed for this gene, rapid detection of five pathogenic Vibrio species in aquatic animals can be achieved; except for Vibrio harveyi, there are two pairs of specific primers for the other 4 pathogenic Vibrio species, which can achieve the purpose of double verification during detection and effectively avoid false negatives caused by gene deletion;

[0021] (2) The detection method provided by the present invention has strong specificity; short detection time; simple and easy to operate; low detection cost; low minimum detection limit, and the minimum detection limits of 9 pairs of specific primers are all between 0.0157 ng / μL - 6.1813 ng / μL; it is suitable for large-scale daily detection in grass-roots farms. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments of the present invention. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1It is the result of agarose gel electrophoresis of PCR products in Example 3 of the present invention. Among them, part A is the optimized result of PCR amplification of the specific primer pair VH-1833-F / R of Vibrio harveyi at gradient annealing temperatures (53-63°C); part B1 is the PCR amplification result of the specific primer pair VP-1833-F / R of Vibrio parahaemolyticus at gradient annealing temperatures (53-63°C); part B2 is the PCR amplification result of the specific primer pair VP-2055-F / R of Vibrio parahaemolyticus at gradient annealing temperatures (53-63°C); part C1 is the PCR amplification result of the specific primer pair VV-1599-F / R of Vibrio vulnificus at gradient annealing temperatures (53-63°C); part C2 is the PCR amplification result of the specific primer pair VV-1776-F / R of Vibrio vulnificus at gradient annealing temperatures (53-63°C); part D1 is the PCR amplification result of the specific primer pair VC-1833-F / R of Vibrio cholerae at gradient annealing temperatures (53-63°C); part D2 is the PCR amplification result of the specific primer pair VC-2055-F / R of Vibrio cholerae at gradient annealing temperatures (53-63°C); part E1 is the PCR amplification result of the specific primer pair VN-1833-F / R of Vibrio anguillarum at gradient annealing temperatures (53-63°C); part E2 is the PCR amplification result of the specific primer pair VN-2055-F / R of Vibrio anguillarum at gradient annealing temperatures (53-63°C). M in the figure is D2000 Marker, and NC1, NC2, NC3, NC4, NC5 and NC6 respectively correspond to the negative controls at annealing temperatures of 53°C, 55°C, 57°C, 59°C, 61°C and 63°C. Lanes 1, 2, 3, 4, 5 and 6 respectively correspond to the PCR amplification products using Vibrio genomic DNA as the template at annealing temperatures of 53°C, 55°C, 57°C, 59°C, 61°C and 63°C.

[0024] Figure 2 It is the result of agarose gel electrophoresis detection of PCR products in Example 4 of the present invention. Among them, part A is the electrophoresis result of PCR products of the specific primer pair VH-1821-F / R for the genomic DNA of Vibrio harveyi and 11 reference bacteria. Lanes 1-12 are the electrophoresis results of PCR products of Vibrio harveyi, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, Vibrio anguillarum, Vibrio alginolyticus, Bacillus subtilis, Bacillus megaterium, Lactobacillus acidophilus, Streptomyces, Pseudomonas pseudoalteromonas and Microbacterium mexicanum in sequence.

[0025] Part B1 shows the PCR amplification results of genomic DNA of Vibrio parahaemolyticus and 11 reference bacteria with the specific primer pair VP-1539-F / R; Part B2 shows the PCR amplification results of genomic DNA of Vibrio parahaemolyticus and 11 reference bacteria with the specific primer pair VP-2040-F / R; Lanes 1-12 are the electrophoresis results of PCR products of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus, Vibrio cholerae, Vibrio anguillarum, Vibrio alginolyticus, Bacillus subtilis, Bacillus megaterium, Lactobacillus acidophilus, Streptomyces, Pseudomonas alteromonas, and Microbacterium mexicanum in sequence;

[0026] Part C1 shows the PCR amplification results of genomic DNA of Vibrio vulnificus and 11 reference bacteria with the specific primer pair VV-1599-F / R; Part C2 shows the PCR amplification results of genomic DNA of Vibrio vulnificus and 11 reference bacteria with the specific primer pair VV-1776-F / R; Lanes 1-12 are the electrophoresis results of PCR products of Vibrio vulnificus, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio cholerae, Vibrio anguillarum, Vibrio alginolyticus, Bacillus subtilis, Bacillus megaterium, Lactobacillus acidophilus, Streptomyces, Pseudomonas alteromonas, and Microbacterium mexicanum in sequence;

[0027] Part D1 shows the PCR amplification results of genomic DNA of Vibrio cholerae and 11 reference bacteria with the specific primer pair VC-1833-F / R; Part D2 shows the PCR amplification results of genomic DNA of Vibrio cholerae and 11 reference bacteria with the specific primer pair VC-2055-F / R; Lanes 1-12 are the electrophoresis results of PCR products of Vibrio cholerae, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio anguillarum, Vibrio alginolyticus, Bacillus subtilis, Bacillus megaterium, Lactobacillus acidophilus, Streptomyces, Pseudomonas alteromonas, and Microbacterium mexicanum in sequence;

[0028] Part E1 shows the PCR amplification results of genomic DNA of Vibrio anguillarum and 11 reference bacteria with the specific primer pair VN-1491-F / R; Part E2 shows the PCR amplification results of genomic DNA of Vibrio anguillarum and 11 reference bacteria with the specific primer pair VN-1833-F / R; Lanes 1-12 are the electrophoresis results of PCR products of Vibrio harveyi, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, Vibrio anguillarum, Vibrio alginolyticus, Bacillus subtilis, Bacillus megaterium, Lactobacillus acidophilus, Streptomyces, Pseudomonas alteromonas, and Microbacterium mexicanum in sequence;

[0029] In each figure, M is D2000 Marker, NC is negative control, and PC is positive control;

[0030] Figure 3It is the detection result of PCR product agarose gel electrophoresis in Example 5 of the present invention. Among them, part A is the electrophoresis result of the PCR product of Vibrio harveyi genomic DNA at 8 dilution gradient concentrations with the primer pair VH-1821-F / R. The electrophoresis results of the PCR products in lanes 1-8 are the electrophoresis results of the PCR products with concentrations of 16.1 ng / μL, 4.025 ng / μL, 1.0063 ng / μL, 0.2516 ng / μL, 0.0629 ng / μL, 0.0157 ng / μL, 0.0039 ng / μL, and 0.0010 ng / μL in sequence;

[0031] Part B1 is the electrophoresis result of the PCR product of Vibrio parahaemolyticus genomic DNA at 8 dilution gradients with the primer pair VP-1539-F / R; Part B2 is the electrophoresis result of the PCR product of Vibrio parahaemolyticus genomic DNA at 8 dilution gradients with the primer pair VP-2040-F / R; The electrophoresis results of the PCR products in lanes 1-8 correspond to the electrophoresis results of the PCR products with concentrations of 24.725 ng / μL, 6.1813 ng / μL, 1.5453 ng / μL, 0.3863 ng / μL, 0.0966 ng / μL, 0.0242 ng / μL, 0.0060 ng / μL, and 0.0015 ng / μL in sequence;

[0032] Part C1 is the electrophoresis result of the PCR product of Vibrio vulnificus genomic DNA at 8 dilution gradients with the primer pair VV-1599-F / R; Part C2 is the electrophoresis result of the PCR product of Vibrio vulnificus genomic DNA at 8 dilution gradients with the primer pair VV-1776-F / R; The electrophoresis results of the PCR products in lanes 1-8 correspond to the electrophoresis results of the PCR products with concentrations of 14.5 ng / μL, 3.625 ng / μL, 0.9063 ng / μL, 0.2266 ng / μL, 0.0566 ng / μL, 0.0142 ng / μL, 0.0035 ng / μL, and 0.0009 ng / μL in sequence;

[0033] Part D1 is the electrophoresis result of the PCR product of Vibrio cholerae DNA concentration at 8 dilution gradients with the primer pair VC-1833-F / R; Part D2 is the electrophoresis result of the PCR product of Vibrio cholerae DNA concentration at 8 dilution gradients with the primer pair VC-2055-F / R; The electrophoresis results of the PCR products in lanes 1-8 correspond to the electrophoresis results of the PCR products with concentrations of 11.325 ng / μL, 2.8313 ng / μL, 0.7078 ng / μL, 0.1770 ng / μL, 0.0442 ng / μL, 0.0111 ng / μL, 0.0028 ng / μL, and 0.0007 ng / μL in sequence;

[0034] Part E1 shows the PCR product electrophoresis results of Vibrio anguillarum DNA concentrations at 8 dilution gradients using the primer pair VN-1491-F / R; Part E2 shows the PCR product electrophoresis results of Vibrio anguillarum DNA concentrations at 8 dilution gradients using the primer pair VN-1833-F / R; Lanes 1-8 correspond to the PCR product electrophoresis results of 7.35 ng / μL, 1.8375 ng / μL, 0.4594 ng / μL, 0.1148 ng / μL, 0.0287 ng / μL, 0.0072 ng / μL, 0.0018 ng / μL, and 0.0005 ng / μL in sequence.

[0035] In each figure, M is D2000 Marker and NC is the negative control.

[0036] Figure 4 It is the agarose gel electrophoresis detection result of the PCR product of a strain of bacteria that can amplify bands in Application Example 1 of the present invention. Among them, lanes 1-9 are the amplification results using the primer pairs VC-1833-F / R, VC-2055-F / R, VV-1599-F / R, VV-1776-F / R, VP-1539-F / R, VP-2040-F / R, VN-1491-F / R, VN-1833-F / R, and VH-1821-F / R in sequence. Detailed implementation manners

[0037] The technical solutions of the present invention will be further described below with reference to the drawings and examples.

[0038] In order to make the purpose, technical solutions and advantages of this application clearer, more thorough and complete, the technical solutions of the present invention will be described clearly and completely below through the drawings and examples. The following detailed descriptions are all descriptions of examples, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.

[0039] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels. Among them, the dry heater was purchased from Hangzhou Qiwei Instrument Co., Ltd.; the Vibrio parahaemolyticus strain 2FRH001 was from the Aquatic Animal Hospital of Dalian Ocean University, the Vibrio alginolyticus strain 2RZH005 was from the Aquatic Animal Hospital of Dalian Ocean University, the Vibrio anguillarum strain 2MHG001 was from the Aquatic Animal Hospital of Dalian Ocean University, the Vibrio harveyi strain ATCC33842 was purchased from the Guangdong Provincial Microbial Culture Collection Center, the Exiguobacterium mexicanum strain D-1 was purchased from the Beijing Biological Collection Center, the Pseudoalteromonas strain C1 was a strain preserved in the laboratory (Xie Xiaochen, Wang Bo, Jiang Jialin, et al. Isolation, identification and application effect analysis of a potential probiotic from the intestine of Ruditapes philippinarum [J]. Journal of Dalian Ocean University, 2024, 39(04): 568-577. DOI: 10.16535 / j.cnki.dlhyxb.2023-259.), the Vibrio vulnificus strain MCCC 1A08743 and the Vibrio cholerae MCCC 1A02608 were purchased from the Marine Microbial Culture Collection Center, the Bacillus subtilis strain ATCC6633 and the Bacillus megaterium strain ATCC14581 were purchased from the Shanghai Microbial Culture Collection Center, the Lactobacillus acidophilus strain BNCC336636 and the Streptomyces strain BNCC335894 were purchased from the Henan Engineering Technology Research Center for Industrial Microbial Strains.

[0040] The 2216E liquid medium consists of the following components: peptone 5.0 g / L, yeast powder 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, sodium nitrate 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L.

[0041] The TCBS agar medium consists of the following components: yeast extract powder 5.0 g / L, peptone 10.0 g / L, sodium thiosulfate 10.0 g / L, sodium citrate 10.0 g / L, oxgall powder 5.0 g / L, sodium taurocholate 3.0 g / L, sucrose 20.0 g / L, sodium chloride 10.0 g / L, ferric citrate 1.0 g / L, bromothymol blue 0.04 g / L, agar 15.0 g / L.

[0042] The information of the reagents (kits) used in the examples is shown in Table 1 below.

[0043] Table 1

[0044] Reagent Source Bacterial Genomic DNA Extraction Kit Tiangen Biochemical Technology (Beijing) Co., Ltd. Primer Pair Beijing Liuhua Huada Gene Technology Co., Ltd. PCR Premix Reagent (2×Rapid Taq Master Mix) Nanjing Novoprotein Scientific Inc. Sterile Ultra-pure Water Nanjing Novoprotein Scientific Inc.

[0045] Example 1

[0046] Design specific primers for five pathogenic Vibrios, the method is as follows:

[0047] Based on the coding genes of bifunctional diguanylate cyclase / phosphodiesterase in the whole genomes of Vibrio harveyi strain SB1, Vibrio cholerae strain RFB16, Vibrio vulnificus strain ATCC 27562, Vibrio anguillarum strain PF4-E1-1, and Vibrio parahaemolyticus strain RIMD2210633 in NCBI, primer design was carried out.

[0048] First, perform BLASTn searches on the coding genes of bifunctional diguanylate cyclase / phosphodiesterase of the five Vibrio strains, download all homologous gene sequences and similar gene sequences, use DNAMAN9 for sequence analysis and alignment, and find the coding genes of specific bifunctional diguanylate cyclase / phosphodiesterase of the same Vibrio species. A total of 9 coding genes of specific bifunctional diguanylate cyclase / phosphodiesterase were found for 5 pathogenic Vibrios, as shown in Table 2. Primer design was carried out based on the specific regions in the specific genes (two pairs of specific primers were designed respectively based on the specific regions of the two bifunctional enzyme coding genes of each Vibrio). A total of 9 pairs of primers were designed, and the primer sequence information and target band sizes are shown in Table 2 and Table 3.

[0049] The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 83584901 is shown in SEQ ID NO.1; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 83584901 is shown in SEQ ID NO.1; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 69719433 is shown in SEQ ID NO.2; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 69720591 is shown in SEQ ID NO.3; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 93897905 is shown in SEQ ID NO.4; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 93897790 is shown in SEQ ID NO.5; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 83860477 is shown in SEQ ID NO.6; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 83858313 is shown in SEQ ID NO.7; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 1191298 is shown in SEQ ID NO.8; The CDS sequence of the bifunctional diguanylate cyclase / phosphodiesterase gene with the gene accession number 1189959 is shown in SEQ ID NO.9.

[0050] SEQ ID NO.1:

[0051]

[0052] SEQ ID NO.2:

[0053]

[0054] SEQ ID NO.3:

[0055]

[0056] SEQ ID NO.4:

[0057]

[0058] SEQ ID NO.5:

[0059]

[0060] SEQ ID NO.6:

[0061]

[0062] SEQ ID NO.7:

[0063]

[0064] SEQ ID NO.8:

[0065]

[0066] SEQ ID NO.9:

[0067]

[0068] Table 2

[0069]

[0070]

[0071] Table 3

[0072]

[0073]

[0074] Example 2

[0075] Extract the DNA of each bacterium as follows:

[0076] Extract the DNA of Vibrio harveyi strain ATCC33842, Vibrio cholerae strain MCCC 1A02608, Vibrio vulnificus strain MCCC1A08743, Vibrio anguillarum strain 2MHG001, Vibrio parahaemolyticus strain 2FRH001, Vibrio alginolyticus strain 2RZH005, Bacillus subtilis strain ATCC6633, Bacillus megaterium strain ATCC14581, Streptomyces strain BNCC335894, Lactobacillus acidophilus strain BNCC336636, Pseudoalteromonas strain C1, and Exiguobacterium mexicanum strain D1.

[0077] First, take the bacterial liquid (containing 25% glycerol) of each bacterium frozen at -80 °C. Dip a small amount of the bacterial liquid with an inoculation loop and streak it onto a 2216E agar plate, and perform activation culture at 30 °C. When single colonies grow out, pick the single colonies and inoculate them into the sterilized and cooled 2216E liquid medium. Culture at 120 rpm and 30 °C until the solution becomes turbid (about 2 days). Then, use a bacterial genomic DNA extraction kit to extract the genomic DNA of these 12 bacteria according to the attached instructions. Use a NanoDrop 2000c micro-spectrophotometer (Thermo Fisher Scientific, USA) to detect the concentration of the obtained genomic DNA. The genomic DNA concentrations of Vibrio harveyi strain ATCC33842, Vibrio cholerae strain MCCC 1A02608, Vibrio vulnificus strain MCCC 1A08743, Vibrio anguillarum strain 2MHG001, Vibrio parahaemolyticus strain 2FRH001, Vibrio alginolyticus strain 2RZH005, Bacillus subtilis strain ATCC6633, Bacillus megaterium strain ATCC14581, Streptomyces strain BNCC335894, Lactobacillus acidophilus strain BNCC336636, Pseudoalteromonas strain C1, and Exiguobacterium mexicanum strain D1 are 64.4 ng / μL, 45.3 ng / μL, 58.0 ng / μL, 29.4 ng / μL, 98.9 ng / μL, 38.8 ng / μL, 10.5 ng / μL, 33.4 ng / μL, 10.3 ng / μL, 21.7 ng / μL, 68.9 ng / μL, and 17.3 ng / μL, respectively.

[0078] Example 3

[0079] Investigate the optimal annealing temperature of the primers designed in Example 1, specifically as follows:

[0080] Using the genomic DNA of Vibrio harveyi strain ATCC33842, Vibrio cholerae strain MCCC 1A02608, Vibrio vulnificus strain MCCC 1A08743, Vibrio anguillarum strain 2MHG001, and Vibrio parahaemolyticus strain 2FRH001 obtained in Example 2 as templates, PCR amplification was performed using the primer pairs designed in Example 1. The annealing temperature in the PCR reaction program was set as gradient temperatures, and the temperature gradient was 53°C, 55°C, 57°C, 59°C, 61°C, and 63°C. To achieve the best results, the primer concentration and PCR extension time used were referred to the optimal reaction conditions described in the instruction manual of the PCR premix reagent (2×Rapid Taq Master Mix), that is, the primer concentration was 10 μmol / L, the PCR extension time was 2 seconds, and the number of PCR cycles was 31 cycles. The PCR reaction program is shown in Table 4. The PCR reaction system was: 0.8 μL of genomic DNA template, 0.8 μL of each upstream and downstream primer, 10 μL of 2×Rapid Taq Master Mix, and sterile ultrapure water was added to 20 μL.

[0081] Table 4

[0082]

[0083] Take 7 μL of the PCR product for a 2% agarose gel electrophoresis experiment, and the agarose gel electrophoresis time was 25 min. The PCR system without adding template DNA was used as a negative control (NC).

[0084] The results are as Figure 1 shown. Only the PCR products with added template DNA showed the target bands, and no bands were run out in the negative controls at each temperature, indicating that the 9 pairs of primers had good specificity and there was no non-specific amplification. The results showed that the primer pair VV-1599-F / R could stably produce a single target band of approximately 521 bp within the annealing temperature range of 53 - 63°C; the primer pairs VV-1599-F / R and VV-1776-F / R could stably produce single target bands of approximately 699 bp and 649 bp respectively within the annealing temperature range of 53 - 63°C; the primer pairs VV-1599-F / R and VV-1776-F / R could stably produce single target bands of approximately 450 bp and 569 bp respectively within the annealing temperature range of 53 - 63°C; the primer pairs VV-1599-F / R and VV-1776-F / R could stably produce single target bands of approximately 397 bp and 208 bp respectively within the annealing temperature range of 53 - 63°C; the primer pairs VV-1599-F / R and VV-1776-F / R could stably produce single target bands of approximately 323 bp and 853 bp respectively within the annealing temperature range of 53 - 63°C.

[0085] The total PCR reaction volume was increased to 40 μL, i.e., 2× Rapid Taq Master Mix 20 μL, forward primer and reverse primer 1.6 μL each, template DNA 1.6 μL, and ddH2O was added to make up to 40 μL. The PCR reaction procedure was referred to Table 4, except that the annealing temperature was selected to be 61°C. The experiment was repeated and the PCR product was sent to Beijing Liuhe BGI Technology Co., Ltd. for first-generation sequencing to further verify whether it was effectively amplified. The sequencing results showed that the consistency of all amplified products with the target fragment could reach more than 99%, indicating that the amplification was effective.

[0086] Embodiment 4

[0087] The optimal annealing temperature of the primers designed in Example 1 was investigated, as follows:

[0088] The DNA of the 12 bacterial samples obtained in Example 2 was used as a template, and the primer pairs designed in Example 1 were used for PCR amplification. In order to enhance the specificity of the 9 pairs of primers and obtain the optimal reaction conditions of the detection method, the number of PCR program cycles was set to 25 cycles, and the annealing temperature of the PCR reaction program of Vibrio harveyi ATCC33842, Vibrio cholerae strain MCCC 1A02608, Vibrio vulnificus strain MCCC 1A08743, Vibrio anguillarum strain 2MHG001, and Vibrio parahaemolyticus strain 2FRH001 was set to 61±0.5°C, and the annealing temperature of the PCR reaction program of Vibrio alginolyticus strain 2RZH005, Bacillus subtilis strain ATCC6633, Bacillus megaterium strain ATCC14581, Streptomyces strain BNCC335894, Lactobacillus acidophilus strain BNCC336636, Pseudomonas strain C1, and Exiguobacterium mexicana strain D1 was set to 61±0.5°C, and the PCR reaction program was shown in Table 5. The PCR reaction system was as follows: 0.8 μL DNA template, 0.8 μL upstream and downstream primers, 10 μL 2× Rapid Taq Master Mix, and sterile ultrapure water to 20 μL.

[0089] Table 5

[0090]

[0091] 7 μL of PCR product was subjected to 2% agarose gel electrophoresis for 25 min. The unused PCR system was used as the positive control (PC), and NC was used as the negative control without template.

[0092] The results are as follows Figure 2As shown, the positive controls in each figure produced the target bands, and each primer pair was successfully amplified to obtain the target bands of the corresponding strains. However, no bands were produced by the detection method using the template DNA of other reference strains, further indicating that the 9 pairs of primers have good specificity.

[0093] Example Five

[0094] Examine the lowest detection limit of the primers designed in Example One, as follows:

[0095] Respectively use the genomic DNAs of Vibrio harveyi, Vibrio cholerae, Vibrio vulnificus, Vibrio anguillarum, and Vibrio parahaemolyticus obtained in Example Two, with concentrations of 64.4 ng / μL, 45.3 ng / μL, 58.0 ng / μL, 29.4 ng / μL, and 98.9 ng / μL respectively. Perform 2-fold serial dilutions with sterile ultrapure water, and the dilution multiples are 2, 4, 8, 16, 32, 64, 128, and 256 times respectively. Take DNA solutions with different dilution multiples as templates and perform PCR amplification with the primer pairs designed in Example One.

[0096] The PCR reaction system is as follows: 0.8 μL of DNA template, 0.8 μL of each upstream and downstream primer, 10 μL of 2×RapidTaq Master Mix, add sterile ultrapure water to 20 μL, and the PCR reaction program refers to Table 5.

[0097] Take 6 μL of the PCR product for 2% agarose gel electrophoresis experiment, and the agarose gel electrophoresis time is 25 min. Use the PCR system without adding template DNA as the negative control (NC).

[0098] The results are as Figure 3 shown. No bands were produced by the negative controls in each figure. In the 20 μL PCR reaction system, the lowest detection limits of the primer pairs VH-1881-F / R, VP-1539-F / R, VP-2040-F / R, VV-1599-F / R, VV-1776-F / R, VC-1833-F / R, and VN-2055-F / R are 0.0157 ng / μL, 6.1813 ng / μL, 6.1813 ng / μL, 0.02266 ng / μL, 0.9063 ng / μL, 0.1700 ng / μL, 0.1700 ng / μL, 0.4594 ng / μL, and 0.1148 ng / μL respectively.

[0099] Application Example One

[0100] Use the 9 pairs of primers designed in Example One to detect 4 bacterial strains isolated from the aquaculture water environment of a seedling breeding workshop of Ruditapes philippinarum in Dalian Donggang. The method is as follows:

[0101] Dilute the aquaculture water in a certain seedling breeding workshop of Ruditapes philippinarum in Donggang, Dalian with sterilized seawater by 10 -3 times. Dip a small amount of the diluted aquaculture water with an inoculation loop and streak it onto a TCBS agar plate. Pick four single colonies with different colony morphologies with an inoculation loop and transfer them into a PCR tube containing 10 μL of sterilized ultrapure water. Place the PCR tube in a dry heater and heat it at a constant temperature of 100 °C for 10 min.

[0102] Using the obtained heated solution as a template, perform PCR amplification with the primer pairs designed in Example 1. The PCR reaction system is as follows: 0.8 μL of template, 0.8 μL of each upstream and downstream primer, 10 μL of 2×Rapid Taq Master Mix, add sterile ultrapure water to 20 μL. The PCR reaction procedure refers to Table 5. Take 6 μL of the PCR product for a 2% agarose gel electrophoresis experiment. The agarose gel electrophoresis time is 25 min. The results of the agarose gel electrophoresis experiment show that the PCR products amplified by VP-1539-F / R and VP-2040-F / R ran out two bright bands, and the band sizes are consistent with the target band sizes. As Figure 4 shown, it can be judged that this strain of bacteria is Vibrio parahaemolyticus. No target products of the other three strains of bacteria were amplified by the 9 pairs of primers, so no bands ran out.

[0103] Amplify the 16S rRNA coding genes of the four strains of bacteria with the forward primer 27F (the sequence is shown in SEQ ID NO.28) and the reverse primer 1492R (the sequence is shown in SEQ ID NO.29). The PCR reaction system is as follows: 1.2 μL of DNA template, 1.2 μL of each upstream and downstream primer, 15 μL of 2×Rapid Taq Master Mix, add sterile ultrapure water to 30 μL.

[0104] The PCR reaction procedure is: 95 °C for 3 min; 95 °C for 15 s, 55 °C for 15 s, 72 °C for 15 s, 31 cycles; 72 °C for extension for 5 min; keep at 4 °C for incubation.

[0105] SEQ ID NO.28: AGAGTTGATCCTGGCTCAG

[0106] SEQ ID NO.29: GGTTACCTTGTTACGACTT

[0107] Take 2 μL of the PCR product for 1.5% agarose gel electrophoresis experiment, and the agarose gel electrophoresis time is 20 min. After electrophoresis is completed, a bright band can be observed by putting it into a gel imager. The successfully amplified PCR product is sent to Beijing Liuhe Huada Gene Technology Co., Ltd. for first-generation sequencing to verify the taxonomic status of the four strains. The 16S amplification sequencing results show that the four strains are Vibrio parahaemolyticus (99% identity with Vibrio parahaemolyticus strain Colony266), Vibrio alginolyticus (99.93% identity with Vibrio alginolyticus strain 2-24), Bacteroides italicus (99.49% identity with Bacteroides italicus strain T38), and Vibrio mediterranei (99.86% identity with Vibrio mediterranei strain 2-40).

[0108] Therefore, the present invention first discovers the coding gene of the bifunctional diguanylate cyclase / phosphodiesterase, which is specific among five pathogenic Vibrio species in aquatic animals. By using the specific primers designed for this gene, rapid detection of five pathogenic Vibrio species in aquatic animals can be achieved; double verification can be achieved during detection, and false negatives caused by gene deletion can be effectively avoided; the provided method has strong specificity; the detection time is short; it is simple and easy to operate; the detection cost is low; the minimum detection limit is low, which is suitable for large-scale daily detection in grass-roots farms.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A biomarker for detecting five pathogenic vibrios of aquatic animals, characterized in that, The five kinds of pathogenic Vibrio for aquatic animals are Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio anguillarum, and Vibrio harveyi; The biomarker is the coding gene of bifunctional diguanylate cyclase / phosphodiesterase, and the CDS sequences of the coding genes of the specific bifunctional diguanylate cyclase / phosphodiesterase of the 5 kinds of pathogenic Vibrio are shown in SEQ ID NO.1 to SEQ ID NO.9 respectively.

2. A marker for detecting pathogenic Vibrio in five aquatic animals according to claim 1, characterized in that: The specific primers for the coding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio parahaemolyticus are VP-1539-F, VP-1539-R, and VP-2040-F, VP-2040-R; the amplification fragment length of the primer pair VP-1539-F and VP-1539-R is 699bp; the amplification fragment length of the primer pair VP-2040-F and VP-2040-R is 649bp; the sequence of VP-1539-F is shown in SEQ ID NO.10, the sequence of VP-1539-R is shown in SEQ ID NO.11, the sequence of VP-2040-F is shown in SEQ ID NO.12, and the sequence of VP-2040-R is shown in SEQ ID NO.

13.

3. A marker for detecting five pathogenic vibrios of aquatic animals according to claim 1, characterized in that: The specific primers for the coding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio cholerae are VC-1833-F, VC-1833-R, and VC-2055-F, VC-2055-R; the amplification fragment length of the primer pair VC-1833-F and VC-1833-R is 397bp; the amplification fragment length of the primer pair VC-2055-F and VC-2055-R is 208bp; the sequence of VC-1833-F is shown in SEQ ID NO.14, the sequence of VC-1833-R is shown in SEQ ID NO.15, the sequence of VC-2055-F is shown in SEQ ID NO.16, and the sequence of VC-2055-R is shown in SEQ ID NO.

17.

4. A marker for detecting five pathogenic vibrios of aquatic animals according to claim 1, characterized in that: The specific primers for the coding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio vulnificus are VV-1599-F, VV-1599-R, and VV-1776-F, VV-1776-R; the amplification fragment length of the primer pair VV-1599-F and VV-1599-R is 455bp; the amplification fragment length of the primer pair VV-1776-F and VV-1776-R is 569bp; the sequence of VV-1599-F is shown in SEQ ID NO.18, the sequence of VV-1599-R is shown in SEQ ID NO.19, the sequence of VV-1776-F is shown in SEQ ID NO.20, and the sequence of VV-1776-R is shown in SEQ ID NO.

21.

5. A marker for detecting pathogenic Vibrio in five aquatic animals according to claim 1, characterized in that: The specific primers for the encoding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio anguillarum are VN-1491-F, VN-1491-R and VN-1833-F, VN-1833-R; the amplification fragment length of the primer pair VN-1491-F and VN-1491-R is 323 bp; the amplification fragment length of the primer pair VN-1833-F and VN-1833-R is 853 bp; the sequence of VN-1491-F is as shown in SEQ ID NO.22, the sequence of VN-1491-R is as shown in SEQ ID NO.23, the sequence of VN-1833-F is as shown in SEQ ID NO.24, and the sequence of VN-1833-R is as shown in SEQ ID NO.

25.

6. A marker for detecting pathogenic vibrios of five aquatic animals according to claim 1, characterized in that: The specific primers for the encoding gene of bifunctional diguanylate cyclase / phosphodiesterase in Vibrio harveyi are VH-1821-F, VH-1821-R; the amplification fragment length of the primer pair VH-1821-F and VH-1821-R is 521 bp; the sequence of VH-1821-F is as shown in SEQ ID NO.26, and the sequence of VH-1821-R is as shown in SEQ ID NO.

27.

7. A method for detecting five pathogenic vibrios of aquatic animals, characterized in that, Using the marker for detecting five kinds of pathogenic Vibrio in aquatic animals according to any one of claims 1 to 6, the method comprises the following steps: Adding the bacteria to be tested into a dry heater, heating at 100 °C for 10 min, using the obtained solution as a template for PCR to detect whether the encoding gene of bifunctional diguanylate cyclase / phosphodiesterase exists. If the encoding gene of bifunctional diguanylate cyclase / phosphodiesterase exists, it indicates that the test substance is one or several of the five kinds of pathogenic Vibrio in aquatic animals; if the encoding gene of bifunctional diguanylate cyclase / phosphodiesterase does not exist, it indicates that the test substance does not contain the five kinds of pathogenic Vibrio in aquatic animals.

8. A method for detecting five pathogenic Vibrio species in aquatic animals according to claim 7, characterized in that, The PCR system is: 0.8 μL of template, 0.8 μL of each upstream and downstream primer, 10 μL of 2×Rapid Taq Master Mix, and adding sterile ultrapure water to 20 μL; The PCR amplification program is: 95 °C, 3 min; 95 °C, 15 s, 61 ± 0.5 °C, 15 s, 72 °C, 2 s, 25 cycles; 72 °C, 5 min; keep at 4 °C.

9. Use of the marker for detecting five kinds of pathogenic Vibrio in aquatic animals according to any one of claims 1 to 6 in the detection of pathogenic Vibrio.

10. A kit for simultaneously detecting five kinds of pathogenic vibrios of aquatic animals, characterized in that: The kit comprises the marker for detecting five kinds of pathogenic Vibrio in aquatic animals according to any one of claims 1 to 6; the five kinds of pathogenic Vibrio in aquatic animals are Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio anguillarum and Vibrio harveyi.

Images