Detection method of highly pathogenic photobacterium damsonii subspecies

Abstract

The invention relates to a detection method of highly pathogenic photobacterium damsonii subspecies, and belongs to the technical field of detection. The Latin name of the high-pathogenicity photobacterium damsela subspecies is Photobacterium damsela subspecies, and the high-pathogenicity photobacterium damsela subspecies contains a characteristic virulence gene, namely, a hemolysin gene sequence; the method comprises the following steps: detecting a hemolysin gene sequence of the highly pathogenic photobacterium damselysin subspecies by using an enzymatic isothermal amplification (ERA) technology; the primer sequence of the ERA is Pdd-F1; r < d-R < 1 >; according to the direction from the 5'tail end to the 3 'tail end, the nucleotide sequence of the Pdd-F1 is a sequence number (ID): 1; the nucleotide sequence of the Pdd-R1 is shown as a sequence number (ID): 2. The detection method provided by the invention has the characteristics of high specificity, high sensitivity, rapidness and convenience.

Description

Technical Field

[0001] The present invention relates to a detection method for Photobacterium damselae subsp. damselae with high pathogenicity, belonging to the technical field of detection. Background Art

[0002] Photobacterium damselae subsp. damselae with high pathogenicity (abbreviated as high-pathogenic PDD) can infect different marine animals such as fish, crustaceans, mollusks, sea turtles, and cetaceans. Photobacterium damselae subsp. damselae (PDD) is widely distributed in the global marine environment and is also pathogenic to humans. The infection of high-pathogenic PDD is typically characterized by hemorrhagic septicemia, with a rapid progression of the disease course and a fatality rate of over 60%. It can cause local hemorrhage in the fin base, eyes, or internal organs of the host, skin ulcerative lesions, and inflammatory reactions in tissues and organs.

[0003] Epidemiological investigations have shown that high-pathogenic PDD exhibits obvious seasonal epidemic characteristics in the cage culture environment, mainly occurring in the high-temperature season, and can trigger an explosive epidemic disease characterized by skin ulcers. This disease is characterized by rapid transmission and extremely high fatality rate. During the disease outbreak, the cumulative mortality rate of a single cage can exceed 60% at most.

[0004] The high-pathogenic strains of PDD contain a virulence plasmid pPHDD1, which encodes a hemolysin damselysin (dly). There is a synergistic effect between dly and the pore-forming toxin hlyApl, which can significantly enhance the hemolytic effect and thus increase the pathogenicity of the bacteria. Existing studies have shown that the dly gene is only present in the high-virulence plasmid of pathogenic PDD and is one of the main causes of host septicemia, skin ulcers, and other diseases. After the high-pathogenic strains lose this gene, they usually transform into low-pathogenic or non-pathogenic strains. Therefore, the dly gene is considered an ideal marker gene for identifying the pathogenicity of Photobacterium damselae subsp. damselae. As a key virulence gene, it deeply affects the pathogenicity of PDD to fish. The high-pathogenic PDD strains carrying the dly gene have an acute onset, a short disease course, and a high fatality rate. They often act together with other pathogens, exacerbating the development of the disease and causing significant economic losses to aquaculture animals. Currently, there is still an insufficient effective treatment method for this disease, and prevention and control still rely on preventive measures and cutting off the transmission route.

[0005] Currently, studies have established conventional PCR and microfluidics-based quantitative real-time PCR (Microfluidics-Based-qRT-PCR) for detecting the dly gene of Photobacterium damselae subsp. piscicida. For the conventional PCR method, only qualitative discrimination of pathogens can be achieved. Limited by the framework of traditional PCR technology, the technical boundary of qualitative detection has not been broken through. At the level of quantitative detection technology, the microfluidics-based quantitative real-time PCR method can achieve precise quantification of pathogen load (the detection limit reaches 1.0×10 1 copies / μL) based on specific primers for the dly gene, but it still relies on expensive professional equipment and cumbersome detection procedures. In addition, the existing technology also has defects such as "not selecting enough pathogens for specific detection", "lacking verification of specific identification of Photobacterium damselae subsp. piscicida (the sequence similarity between PDP and PDD dly genes > 99.4%) and having a risk of false positive reactions caused by high sequence homology, which may lead to clinical misjudgment".

[0006] Enzymatic recombinase amplification (ERA) is a constant-temperature nucleic acid amplification technology, which has been successfully applied in the detection of aquatic pathogens due to its simplicity, rapidity and high efficiency. It breaks through the dependence on large-scale instruments in traditional PCR, realizes the rapid amplification of pathogen nucleic acids under the broad constant-temperature conditions of 25 to 42°C, and the amplification efficiency can reach billions of times within a few minutes. RT-ERA (real-time enzymatic recombinase amplification assay) can quantify pathogens and predict disease risks in a short time. ERA-LFD (ERA combined with lateral flow dipsticks (LFD) assay) shows the advantages of on-site detection without precise temperature control equipment with its rapid visualization of detection results within 10 minutes. There has been no report on the detection of Photobacterium damselae subsp. piscicida by ERA. Summary of the Invention

[0007] The object of the present invention is to provide a method for detecting Photobacterium damselae subsp. piscicida.

[0008] To achieve the object of the present invention, the Latin name of the highly pathogenic Photobacterium damselae subsp. damselae is Photobacterium damselae subsp. damselae, which contains a characteristic virulence gene - the gene sequence of hemolysin damselysin (dly; a hemolysin); the method includes detecting the gene sequence of hemolysin damselysin of the highly pathogenic Photobacterium damselae subsp. damselae by the enzyme - mediated isothermal amplification (ERA) technique; the primer sequences of the ERA are: Pdd - F1; Pdd - R1; in the direction from the 5' end to the 3' end, the nucleotide sequence of Pdd - F1 is sequence number (ID): 1; the nucleotide sequence of Pdd - R1 is sequence number (ID): 2.

[0009] In a specific embodiment, the length of the primer is 236bp.

[0010] In a specific embodiment, the ERA includes real - time fluorescence quantitative combined with ERA or ERA combined with a lateral flow chromatographic test strip.

[0011] In a specific embodiment, the probe of the real - time fluorescence quantitative combined with ERA is Pdd - probe1a. In the direction from the 5' end to the 3' end, the nucleotide sequence of Pdd - probe1a is sequence number (ID): 11. A FAM fluorophore is introduced at the 30th base at the 5' end of sequence number (ID): 11, and a BHQ1 quenching group is designed at the 15th base at the 3' end. A tetrahydrofuran is set between the FAM fluorophore and the BHQ1 quenching group, and a C3 - Spacer is set at the 3' end as a blocking group;

[0012] The probe of the ERA combined with a lateral flow chromatographic test strip is: Pdd - Sprobe1a. In the direction from the 5' end to the 3' end, the nucleotide sequence of Pdd - Sprobe1a is sequence number (ID): 12. A FAM fluorophore is set at the 5' end, a tetrahydrofuran is set at the 30th base at the 5' end, and a C3 - Spacer blocking group is set at the 3' end.

[0013] In a specific embodiment, the length of the probe is 46bp.

[0014] In a specific embodiment, the primer of the ERA combined with a lateral flow chromatographic test strip is Pdd - R1 - biotin, and Pdd - R1 - biotin is labeled with a vitamin B7 at the 5’ end of Pdd - R1.

[0015] In a specific embodiment, the reaction temperature of the real-time fluorescence quantitative combination with ERA is 33°C to 41°C, preferably 39°C.

[0016] In a specific embodiment, the reaction time of the real-time fluorescence quantitative combination with ERA is 10 to 20 min.

[0017] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatographic test strip is 37°C to 41°C, preferably 40°C; the reaction time is 10 to 35 min, and the preferred reaction time is 10 min.

[0018] In a specific embodiment, the lowest detection limit of the ERA is 2×10 0 copies / μL.

[0019] Beneficial effects:

[0020] 1. The present invention adopts specific primers and probes, enabling RT-ERA and ERA-LFD to operate under a constant temperature condition not exceeding 40°C. In particular, RT-ERA can reach the plateau of the reaction within 13 to 20 minutes, while ERA-LFD can complete accurate detection within approximately 10 minutes.

[0021] 2. The RT-ERA method of the present invention realizes the accurate quantitative detection of Photobacterium damselae subsp. piscicida with high pathogenicity by establishing a linear relationship (y = -2.4077x + 25.383, R 2 = 0.9866) between the plasmid copy number and the fluorescence intensity threshold Ct value. Using the plasmid standard as a template, the detection limits of both RT-ERA and ERA-LFD methods are 2.0×10 0 copies / μL.

[0022] 3. In the specificity experiment of the method of the present invention, there is a specific reaction only with Photobacterium damselae subsp. piscicida with high pathogenicity, and there is no cross-reaction with 15 strains of low pathogenicity PDD from other different sources, 3 strains of Photobacterium damselae subsp. piscicida (PDP), and 8 common pathogens of groupers - Amyloodinium ocellatum, Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus iniae, nervous necrosis virus of groupers (VNN), and iridovirus of groupers (SGIV). For 70 grouper tissue samples, 24 Sebastodes fuscens, 12 Penaeus vanname, and 26 aquaculture water samples collected from different regions in Hainan, China, the coincidence rates of the detection results of RT-ERA and ERA-LFD with the basic ERA method are 100%, and the detection sensitivities are higher than those of conventional PCR.

[0023] 4. The method of the present invention does not rely on professional PCR instruments and constant-temperature operation. Its rapid detection and high sensitivity show great potential in the detection of aquatic pathogens. Compared with nucleic acid amplification methods such as conventional PCR, it has the advantages of simple operation, high efficiency, high sensitivity, and strong practicability.

[0024] 5. The RT-ERA and ERA-LFD detection methods established in the present invention are characterized by high specificity, high sensitivity, and rapidity and convenience. They can not only evaluate the pathogen load in fish tissues but also be applied to the detection of pathogens in aquaculture water bodies, which is of great value for the early warning and prevention and control of the highly pathogenic Photobacterium damselae subsp. piscicida. At the same time, it also provides an important technical reference for the detection of other aquatic pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the screening result of the best primers for the highly pathogenic Photobacterium damselae subsp. piscicida. M: Maker D 2000, 1: negative control; 2 - 6 represent primer combinations 1 - 5 in Table 1 respectively;

[0026] Figure 2 It is the detection result of the highly pathogenic Photobacterium damselae subsp. piscicida in Examples 2 - 4. Figure 2 In Figure A of the middle figure is Example 2, Figure B is Example 3, and Figure C is Example 4, NTC: negative control. Each RT-ERA temperature condition was repeated three times. In ERA-LFD, if only a blue control line appears on the test strip, it indicates that the test result is negative; if both a blue control line and a red test line appear on the test strip, it indicates that the test result is positive. The reaction intensity is determined by the depth of the red color of the test line;

[0027] Figure 3 It is the sensitivity test result diagram of Example 5; Figure A is the sensitivity of conventional PCR; Figure B is the sensitivity of ERA; Figure C is the sensitivity test of RT-ERA; Figure D is the standard curve of RT-ERA; Figure E is the sensitivity test of ERA-LFD; NTC: negative control;

[0028] Figure 4 It is the specific detection result of the highly pathogenic Photobacterium damselae subsp. piscicida in Example 6. A: RT-ERA specificity experiment; the amplification curves shown are for 5 strains of highly pathogenic PDD. B: ERA-LFD specificity experiment; those with red bands are positive. 1 - 5 are 5 strains of highly pathogenic PDD from different sources, 6 - 20 are 15 strains of lowly pathogenic PDD from different sources, 21 - 23 are 3 strains of Photobacterium damselae subsp. piscicida from different sources, 24 - 31 are 8 common seawater pathogens such as groupers, and 32 is the negative control;

[0029] Figure 5 Detection results of highly pathogenic PDD in 132 samples collected from farms by the conventional PCR method in Example 7. Among them, 1 to 106 (Table 3 "T1 to T106") respectively represent a total of 106 Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂, Plectropomus leopardus, Sebastes schlegelii, hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂×Epinephelus moara♂) and Litopenaeus vannamei; 107 to 132 (Table 4 "W1 to W26") represent 26 water samples, M is the Marker band, and N is the negative control;

[0030] Figure 6 Detection results of highly pathogenic PDD in 132 samples collected from farms by the basic ERA method in Example 7. Among them, 1 to 106 (Table 3 "T1 to T106") respectively represent a total of 106 Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂, Plectropomus leopardus, Sebastes schlegelii, hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂×Epinephelus moara♂) and Litopenaeus vannamei; 107 to 132 (Table 4 "W1 to W26") represent 26 water samples, M is the Marker band, and N is the negative control;

[0031] Figure 7 Detection results of Photobacterium damselae subsp. piscicida (PDD) in 132 samples collected from farms by the RT-ERA method in Example 7. 30 positive samples were detected and the copy number of the highly pathogenic PDD gene was calculated according to the Ct value;

[0032] Figure 8 Detection results of Photobacterium damselae subsp. piscicida (PDD) in 132 samples collected from farms by the ERA-LFD method; among them, 1 to 106 (Table 3 "T1 to T106") respectively represent a total of 106 Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂, Plectropomus leopardus, Sebastes schlegelii, hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂×Epinephelus moara♂) and Litopenaeus vannamei; 107 to 132 (Table 4 "W1 to W26") represent 26 water samples, and N is the negative control. Detailed implementation manners

[0033] To achieve the object of the present invention, the Latin name of the highly pathogenic Photobacterium damselae subsp. piscicida is Photobacterium damselae subsp. damselae, which contains the characteristic virulence gene - the hemolysin damselysin gene sequence; the method includes detecting the hemolysin damselysin gene sequence of the highly pathogenic Photobacterium damselae subsp. piscicida by the enzyme-mediated isothermal amplification technology ERA; the primer sequences of the ERA are: Pdd-F1; Pdd-R1; in the direction from the 5' end to the 3' end, the nucleotide sequence of Pdd-F1 is the sequence number (ID): 1; the nucleotide sequence of Pdd-R1 is the sequence number (ID): 2.

[0034] In a specific embodiment, the length of the primer is 236 bp.

[0035] In a specific embodiment, the ERA includes real-time fluorescence quantitative binding ERA or ERA binding lateral flow test strip.

[0036] In a specific embodiment, the probe of the real-time fluorescence quantitative binding ERA is Pdd-probe1a. According to the direction from the 5'-end to the 3'-end, the nucleotide sequence of the Pdd-probe1a is the sequence number (ID): 11. A FAM fluorophore is introduced at the 30th base at the 5'-end of the sequence number (ID): 11, and a BHQ1 quenching group is designed at the 15th base at the 3'-end. A tetrahydrofuran is set between the FAM fluorophore and the BHQ1 quenching group, and a C3-Spacer is set at the 3'-end as a blocking group;

[0037] The probe of the ERA binding lateral flow test strip is: Pdd-Sprobe1a. According to the direction from the 5'-end to the 3'-end, the nucleotide sequence of the Pdd-Sprobe1a is the sequence number (ID): 12. A FAM fluorophore is set at the 5'-end of the sequence number (ID): 12, a tetrahydrofuran is set at the 30th base at the 5'-end, and a C3-Spacer blocking group is set at the 3'-end at the same time.

[0038] In a specific embodiment, the length of the probe is 46 bp.

[0039] In a specific embodiment, the primer of the ERA binding lateral flow test strip is Pdd-R1-biotin, and the Pdd-R1-biotin is labeled with a vitamin B7 at the 5’-end of the Pdd-R1.

[0040] In a specific embodiment, the reaction temperature of the real-time fluorescence quantitative binding ERA is 33°C to 41°C, preferably 39°C.

[0041] In a specific embodiment, the reaction time of the real-time fluorescence quantitative binding ERA is 10 to 20 min.

[0042] In a specific embodiment, the reaction temperature of the ERA binding lateral flow test strip is 37°C to 41°C, preferably 40°C; the reaction time is 10 to 35 min, and the preferred reaction time is 10 min.

[0043] In a specific embodiment, the lowest detection limit of the ERA is 2×10 0 copies / μL.

[0044] The specific implementation manners of the present invention will be further described below in conjunction with embodiments, and the present invention is not thereby limited to the scope of the described embodiments.

[0045] 1 Main materials

[0046] The experimental samples include: (1) Photobacterium damselae subsp. damselae (PDD): 5 highly pathogenic strains (3 strains isolated from different regions in Hainan Province; 2 strains provided by Teacher Yu Yongxiang of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, collected from Haiyang County, Shandong Province and Changdao District, Yantai City respectively); 15 low pathogenic strains (9 strains isolated from Hainan Province; 6 strains provided by the Yellow Sea Institute, collected from Haiyang County, Dongying City and Changdao District, Yantai City respectively); (2) Photobacterium damselae subsp. piscicida (PDP): 3 strains isolated from fish farms in Hainan Province; (3) Common pathogens of groupers: Amyloodinium ocellatum, Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus iniae, Viral nervous necrosis (VNN) of groupers, and Singapore grouper iridovirus (SGIV). All the above pathogens are preserved in this laboratory.

[0047] In the clinical experiment, a total of 132 samples were prepared. Among them, 28 tissue samples of Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂ were from a grouper farm in Wenchang City, Hainan Province (2023) of this laboratory; 24 tissue samples of Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂ and Plectropomus leopardus, as well as 26 water samples from cement pond ponds, were collected from Chengmai County, Danzhou City and Lingao County, Hainan Province (2024); In addition, 18 tissue samples of Epinephelus fuscoguttatus♀×Epinephelus tukula♂, 12 tissue samples of Penaeus vannamei, and 24 tissue samples of Sebastes schlegeli were provided by Teacher Yu Yongxiang of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, collected from Haiyang County, Shandong Province (2021), Dongying City and Changdao District, Yantai City (2022) respectively.

[0048] 2 Main reagents

[0049] ERA Basic Nucleic Acid Amplification Kit, ERA Fluorescent Nucleic Acid Amplification Kit, ERA Test Strip Type Nucleic Acid Amplification Kit, Flow-through Test Strip (Suzhou Xianda Company, China), Tissue Genomic DNA Extraction Kit (Omega Bio-Tek Company, USA), Water Body Genomic DNA Extraction Kit (Omega Bio-Tek Company, USA), Super TotalRNAExtraction Kit (LS1040) Total RNA Extraction Kit (Promega (Beijing) Biotechnology Co., Ltd., China), FastPure Gel DNA Extraction Mini Kit DC301-01 Gel Extraction / DNA Purification Kit (Nanjing Novozymes Biotech Co., Ltd., China).

[0050] To avoid false positive results or template contamination in the experiments of the present invention, a partition isolation operation method is adopted. The reagent area, amplification area, and detection area need to be strictly separated. Before leaving the first two areas (reagent area and amplification area), it is necessary to ensure that the test tubes are tightly sealed. When performing the final detection, the test strips with the reaction solution aspirated should not be placed together before they are completely dry.

[0051] Example 1

[0052] 5 primer combinations.

[0053] In the design of the fluorescent probe, a FAM fluorescent group was introduced at the 30th base at the 5' end, and a BHQ1 quenching group was designed at the 15th base at the 3' end. To ensure the effective transmission of the fluorescent signal, a deoxyribonucleotide analogue (tetrahydrofuran, THF) was used to separate between the fluorescent group and the quenching group, and a C3-Spacer was set at the 3' end as a blocking group. For the test strip type probe, a FAM fluorescent group was also set at the 5' end, THF was added at the 30th base at the 5' end, and a C3-Spacer blocking group was equipped at the 3' end. Such design ensures that the total length of both probes is 46 base pairs, optimizing the detection performance. Primer and probe information is shown in Table 1.

[0054] Table 1 Primer and Probe Information

[0055]

[0056] Construction and Synthesis of Positive Plasmid Standard

[0057] Using the positive sample of intestinal tissue infected with highly pathogenic PDD as a template, the dly gene of highly pathogenic PDD was amplified with the primer combination Pdd-F1 / Pdd-R1 (Table 1). The amplified fragment was gel-purified and recovered using the FastPure Gel DNA Extraction Mini Kit DC301-01 (Nanjing Novoprotein Scientific Inc., China). The purified product was ligated into the PEASY-T1 Sample vector CT111 (TransGen Biotech Co., Ltd., China) to construct the recombinant plasmid PEASY-DLY. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells ZC101 (Beijing Zhuangmeng International Biotechnology Co., Ltd., China), and spread on an LB plate containing ampicillin. The cells were cultured in a 37°C incubator until colonies grew. Positive colonies were picked and diluted in enzyme-free and sterile water, screened using M13 vector primers and colony PCR, and identified by sequencing. After the positive clone strains were inoculated into LB liquid medium and cultured overnight, plasmid extraction was performed using the Omega Plasmid Mini Kit I D6943 (Omega Bio-Tek, USA). The concentration of plasmid DNA was determined to be 101 ng / μL by a Shanghai Yidian spectrophotometer, and the plasmid concentration was calculated to be 2.0×10 10 copies / μL according to Avogadro's constant, and stored at -20°C for later use in subsequent ERA detection.

[0058] Template DNA extraction

[0059] Total tissue DNA extraction was performed on the midgut tissue (including intestinal contents) DNA of Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂; the midgut tissue (including intestinal contents) DNA of Plectropomus leopardus; the liver tissue, midgut tissue (including intestinal contents) and skin tissue DNA of Epinephelus fuscoguttatus♀×Epinephelus polyphekadion♂; the liver tissue, midgut tissue (including intestinal contents) and skin tissue DNA of Sebastes schlegelii; the hepatopancreas tissue and intestinal tissue (including intestinal contents) DNA of Litopenaeus vannamei according to the instructions of the OMEGA E.Z.N.A. Tissue DNA Kit D3396. For each sample, about 30 mg was taken for total tissue DNA extraction. Then, the DNA concentration was measured using a Nanodrop micro-spectrophotometer and uniformly controlled at 200 ng / μL (if the concentration was too low, extraction was repeated; if the concentration was too high, it was diluted with Elutionbuffer in the kit). The eluted DNA was stored at -20°C.

[0060] DNA extraction from water samples: First, filter a water body sample (5 L) using a microporous filter paper (pore size 0.22 μm). Cut the filtered filter paper into four pieces and place them in a clean 50 mL centrifuge tube. Then, perform total water sample DNA extraction according to the instructions of the OMEGA E.Z.N.A. Water DNA Kit D5525-01. Measure the DNA concentration using a Nanodrop micro-spectrophotometer and uniformly control it to 50 ng / μL (if the concentration is too low, extract again; if the concentration is too high, dilute it with the Elution buffer in the kit). Store the eluted DNA at -20 °C.

[0061] Basic ERA detection

[0062] For basic ERA, use the GenDx ERA basic nucleic acid amplification kit KS101 (Suzhou Xianda Gene Technology Co., Ltd., China).

[0063] The reaction system of basic ERA consists of 2.5 μL of forward primer (10 μM), 2.5 μL of reverse primer (10 μM), 2 μL of template DNA, 20 μL of solubilizer, and 21 μL of enzyme-free water to form a 48 μL premix. First, add 48 μL of the premix to each tube of amplification reagent. Then, add 2 μL of ERA activator (containing Mg + ) to the tube cap, and centrifuge quickly and mix well. Using a plasmid with a concentration of 2×10 5 copies / μL as the template, first perform basic ERA reaction tests using the 5 different primer combinations in Table 1 under the guiding temperature (41 °C) and time parameters (15 min) provided by the kit.

[0064] RT-ERA detection

[0065] For RT-ERA, use the GenDx ERA fluorescent nucleic acid amplification kit KS103 (Suzhou Xianda Gene Technology Co., Ltd., China).

[0066] The reaction system of RT-ERA consists of 2.1 μL of forward primer (10 μM), 2.1 μL of reverse primer (10 μM), 0.6 μL of probe (10 μM), 2 μL of template DNA, 20 μL of solubilizer, and 21.2 μL of enzyme-free water to form a 48 μL premix. First, add 48 μL of the premix to each tube of amplification reagent. Then, add 2 μL of ERA activator (containing Mg +) Centrifuge quickly and mix well. Detect the fluorescence value using a Q2000B real-time fluorescence quantitative PCR instrument (Hangzhou Langji Technology Instruments Co., Ltd., China). Set the experiment type to absolute quantification, the dye to TaqMan Reagents, and the reaction program to 40 cycles with fluorescence values read every 30 s. The amplification curve of the positive template is S-shaped, while there is no amplification in the negative control. The point where the set threshold (300 CFU) intersects the amplification curve is the Ct value. The slope of the amplification curve in the logarithmic growth phase is the slope of the curve. When the amplification curve enters the plateau phase, the fluorescence intensity corresponding to the curve is the final fluorescence value. Using a plasmid with a concentration of 2×10 5 copies / μL as the template, the primers and probes are as shown in Table 1 above, with the reaction temperature at 39 °C and the reaction time at 20 min.

[0067] ERA-LFD detection

[0068] ERA-LFD uses the GenDx ERA strip nucleic acid amplification kit KS105 and the flow-through test strip TS101 (Suzhou Xianda Gene Technology Co., Ltd., China).

[0069] The primers and probes used in ERA-LFD are shown in Table 1. The reaction system consists of 2.1 μL of forward primer (10 μM), 2.1 μL of reverse primer (10 μM), 0.6 μL of probe (10 μM), 2 μL of template DNA, using a plasmid with a concentration of 2×10 5 copies / μL as the template, 20 μL of solubilizer, and 21.2 μL of enzyme-free water, together forming a 48-μL premix. First, add 48 μL of the premix to each tube of amplification reagent. Then add 2 μL of ERA activator (containing Mg + ) to the tube cap, centrifuge quickly and mix well. Then, terminate the reaction using 2 μL of proteinase K (5 μg / 100 μL) according to the instructions. The primers and probes are as shown in Table 1, with the reaction temperature at 40 °C and the reaction time at 20 min.

[0070] When observing the reaction effect using a test strip, the amplified product needs to be diluted with enzyme-free water at a dilution ratio of 40:1 (i.e., take 5 μl of the reaction product into a 1.5 mL centrifuge tube and add 200 μl of pure water for dilution), then insert the test strip into the centrifuge tube, and read the result after 7 - 10 min. The judgment criteria are as follows: If only a single blue band (control line) appears in the control area, this situation is judged as a negative result; if two bands appear simultaneously on the test strip, one in the control area (control line) and the other in the test area (test line) presenting red, it is judged as a positive result. The reaction intensity is characterized by the color depth of the band in the test area (test line). The darker the red tone of the band in the test area (test line), the higher the reaction intensity.

[0071] Experimental results: All primer combinations listed in Table 1 successfully amplified the target sequence. M: D 2000 Marker, 2 - 6: 1 - 5 primer combinations. On the agarose gel, primer combinations 2, 3, and 4 produced non-specific bands; the band of primer combination 5 was lighter in color, so the above combinations were abandoned. The experimental results are as Figure 1 shown. Primer combination 1 (Pdd-F1 / Pdd-R1), with the amplified target product length of 236 bp, had the best effect.

[0072] Example 2

[0073] For RT-ERA detection, primers Pdd-F1 / Pdd-R1 and probe Pdd-probe1a were used. Using the recombinant plasmid standard with a concentration of 2×10 5 copies / μL as the positive template, reactions were carried out at 33℃, 35℃, 37℃, 39℃, and 41℃ for 20 min respectively, and the reaction system referred to Example 1. The experimental results are as Figure 2 shown in A. At 39℃, the amplification curve showed the lowest cycle threshold (Ct value), the largest slope, and the highest fluorescence intensity. 39℃ was the optimal reaction temperature. In addition, the optimal reaction time range corresponding to the interval (26 - 40 cycles) when the cycle number reached the plateau was 13 - 20 min.

[0074] Example 3

[0075] For ERA-LFD detection, primers Pdd-F1 / Pdd-R1-biotin and probe Pdd-Sprobe1a were used. Similarly, using the recombinant plasmid with a concentration of 2×10 5The plasmid standard at copies / μL was used as the positive template, and the reaction system referred to Example 1. The reaction was carried out at 37°C, 38°C, 39°C, 40°C and 41°C for 20 min respectively. After the reaction, 5 μL of the reaction solution was taken and mixed with 200 μL of enzyme-free water, and detected with a test strip. The experimental results are as Figure 2 shown in B. The control band was the most obvious red at 40°C, and 40°C was used as the optimal reaction temperature.

[0076] Example 4

[0077] For ERA-LFD detection, primers Pdd-F1 / Pdd-R1-biotin and probe Pdd-Sprobe1a were used. Similarly, with the plasmid standard at a concentration of 2×10 5 copies / μL as the positive template, the reaction system referred to Example 1. At 40°C as the reaction temperature, the reaction was carried out for 5 min, 10 min, 15 min, 20 min and 25 min respectively. The results are as Figure 2 shown in C. The band color was very weak at 5 min, and the control band was very obvious red during 10 - 35 min and the color depth did not deepen with the increase of time. Therefore, the optimal color development time was 10 min.

[0078] Example 5

[0079] Sensitivity test

[0080] The recombinant plasmid standard was serially diluted. Using the recombinant plasmid standard at a concentration of 2.0×10 0 ~2.0×10 7 copies / μL as the template, conventional PCR, basic ERA, RT-ERA and ERA-LFD detections were carried out respectively. Conventional PCR: The primers used were Dly-F / Dly-R (Table 1). The reaction system (50 μL) included 2 μM (10 μL) forward primer, 2 μL (10 μL) reverse primer, 2 μL template DNA, 25 μL of 2×Rapid Taq Master Mix and dd water supplemented to 50 μL. DNA denaturation was carried out at 95°C for 5 min, and then a total of 35 PCR cycles were carried out. The specific conditions were as follows: DNA denaturation at 94°C for 30 s, primer annealing at 57°C for 30 s, and DNA polymerization at 72°C for 1 min. After the last cycle, the reaction was terminated at 72°C for 10 min.

[0081] Basic ERA: Pdd-F1 / Pdd-R1 (Table 1), reaction temperature and time: 41°C for 15 min. RT-ERA: Pdd-F1 / Pdd-R1 / Pdd-probe1a (Table 1), reaction temperature and time: 39°C for 13 min. ERA-LFD: Pdd-F1 / Pdd-R1-biotin / Pdd-Sprobe1a (Table 1), reaction temperature and time: 40°C for 10 min. For this sensitivity experiment, the reaction systems of basic ERA, RT-ERA, and ERA-LFD all refer to Example 1.

[0082] The results of conventional PCR detection are as Figure 3 shown in A. Bands were shown in the concentration range of 2.0×10 1 ~2.0×10 7 copies / μL, and the lowest detection limit was 2.0×10 1 copies / μL. The results of basic ERA detection are as Figure 3 shown in B. Bands were shown in the concentration range of 2.0×10 0 ~2.0×10 7 copies / μL, and the lowest detection limit was 2.0×10 0 copies / μL. The results of RT-ERA detection are as Figure 3 shown in C. Amplification curves appeared in three biological replicates of each concentration gradient. Therefore, the lowest detection limit of the RT-ERA method established in this study was 2.0×10 0 copies / μL. According to the relationship between the copy number (x) of the recombinant plasmid and the threshold cycle number Ct value (y), a standard curve was plotted. The formula was y = -2.4077x + 25.383 ( Figure 3 D), and the correlation coefficient R 2 = 0.9866. The results of ERA-LFD detection are as Figure 3 shown in E. Red bands appeared in the test line of the test strip in the concentration range of 2.0×10 0 ~2.0×10 7 copies / μL. Therefore, the lowest detection limit of the ERA-LFD method established in this study was 2.0×10 0 copies / μL.

[0083] Example 6

[0084] Five highly pathogenic PDDs, 15 lowly pathogenic PDDs, 3 PDPs from different sources of known pathogens and strains, as well as Amyloodinium ocellatum, Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus iniae, grouper iridovirus and nervous necrosis virus (cDNA) were standardized to a uniform DNA concentration of 200 ng / μL for standby according to the requirements of the DNA extraction process.

[0085] Using the recombinant plasmid standard with a concentration of 2×10 5 copies / μL as the positive template, RT-ERA used the primer and probe combination of Pdd-F1 / Pdd-R1 / Pdd-probe1a (Table 1), the reaction temperature was 39 °C, and the reaction time was 13 min (refer to Example 3). ERA-LFD used the primer and probe combination of Pdd-F1 / Pdd-R1-biotin / Pdd-Sprobe1a (Table 1), the reaction temperature was 40 °C, and the reaction time was 10 min (refer to Example 4). The reaction system referred to Example 1. Five highly pathogenic PDDs, 15 lowly pathogenic PDDs, 3 PDPs from different sources of known pathogens and strains, as well as Amyloodinium ocellatum, Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus iniae, grouper iridovirus and nervous necrosis virus (cDNA) were detected. The detection results of highly pathogenic PDDs of known pathogens and strains are shown in Table 2 and Figure 4 as follows. In RT-ERA, only the amplification curves appeared in the samples of 5 highly pathogenic PDDs from different sources ( Figure 4 A); in ERA-LFD, only the positive red bands appeared on the test strips corresponding to this sample ( Figure 4 B). The rest of the pathogens and the negative control were all negative results, indicating that the two detection methods established in this study could specifically distinguish pathogens such as lowly pathogenic PDD and Photobacterium damselae subsp. piscicida, with good specificity.

[0086] Table 2 Specific detection results of highly pathogenic Photobacterium damselae subsp. piscicida (PDD)

[0087]

[0088]

[0089] Note: The detection results of RT-ERA and ERA-FLD are consistent. -, representing a negative detection result; +, representing a positive detection result.

[0090] Example 7

[0091] Detection of clinical tissue samples and water samples

[0092] A total of 106 tissue samples (Table 3) and 26 water samples (Table 4) were collected during 2021 - 2024. RT-ERA and ERA-LFD were used to detect highly pathogenic PDD, and the results were compared with those of conventional PCR and basic ERA. A recombinant plasmid standard with a concentration of 2×10 5 copies / μL was used as the positive template. The reaction conditions and system of conventional PCR refer to Example 6. RT-ERA used the primer and probe combination of Pdd-F1 / Pdd-R1 / Pdd-probe1a (Table 1), with a reaction temperature of 39°C and a reaction time of 13 min. ERA-LFD used the primer and probe combination of Pdd-F1 / Pdd-R1-biotin / Pdd-Sprobe1a (Table 1), with a reaction temperature of 40°C and a reaction time of 10 min. The reaction system refers to Example 1.

[0093] According to the method for extracting tissue DNA in Example 1 above, tissue samples with a DNA concentration of 200 ng / μL per sample were prepared. At the same time, according to the experimental settings in Example 1, 2 μL of the template was added. It can be calculated that the DNA content in each DNA sample was 400 ng. Similarly, the DNA concentration of each water sample was 50 ng / μL. After adding 2 μL of the template, the DNA content of each DNA sample was 80 ng.

[0094] The specific gel images, fluorescence amplification curves, and test strip results of each detection method are shown respectively as Figures 5 to 8 follows. Among them, 14 positive samples were detected by the conventional PCR method: the results are shown in Figure 5 A - J, tissue samples: F9P1, F9P2, F9P3, F11P1, F11P5, F11P6, F11P7, F12P10, F12P11 (2 copies), F12P12 (2 copies), water samples: F7P1, F9P3; M is the Marker band, N is the negative control. 30 positive samples were detected by the basic ERA method: the results are shown in Figure 6 A - J;; M is the Marker band, N is the negative control. 30 positive samples were detected by the RT-ERA method and the copy number of the highly pathogenic PDD gene was calculated according to the Ct value: the results are shown in Figure 7 ; 30 positive samples were detected by the ERA-LFD method: the results are shown in Figure 8 A - J, N is the negative control.. 7 samples from Farm F1, F5P1, F6P1, F7P1, F7P2, F7P3, F7P4, F7P5, F9P1, F9P2, F9P3, F11P1, F11P5, F11P6, F11P7, F12P10, F12P11 (2 copies), F12P12 (2 copies), water samples: F5P1, F7P1, F9P1, F9P3.

[0095] Table 3. Detection results of conventional PCR, ERA, RT-ERA and ERA-LFD for clinical fish samples

[0096]

[0097]

[0098]

[0099] -, negative; +, positive.

[0100] Table 4. Detection results of conventional PCR, ERA, RT-ERA and ERA-LFD for environmental water samples

[0101]

[0102]

[0103] -, negative; +, positive.

[0104] The RT-ERA and ERA-LFD detection methods of the present invention have multiple advantages. It not only provides an accurate dly gene quantification scheme for existing detection methods, but also realizes rapid detection with high sensitivity, and the detection limit reaches 2.0×10 0 copies / μL. Among them, compared with fluorescence quantitative PCR, RT-ERA does not require cumbersome temperature change procedures; compared with the conventional PCR-based gel electrophoresis detection method, ERA-LFD has greater convenience.

[0105] The RT-ERA reaction of the present invention is effective in the temperature range of 33-41°C. The Ct value is the lowest, the slope is the highest and the fluorescence value is the largest at 39°C. The reaction reaches the plateau within 26-40 cycles (13-20 min). According to the brightness of the control band in red, it is found that the optimal reaction temperature and time of ERA-LFD are 40°C and 10 min respectively. RT-ERA combined with a portable fluorescence quantitative analyzer can accurately judge the result through a quantitative curve at 39°C within 13-20 min. The result interpretation of ERA-LFD is clear, and the result can be obtained at 40°C within 10 min, which is more convenient, clear and intuitive than the result interpretation based on the bands on the gel map of basic ERA and PCR. In addition, in order to avoid false positives or template contamination, partition isolation operation is adopted. This method helps to improve the accuracy of our ERA detection method.

[0106] The present invention optimizes the content of specific detection, and adds multiple categories and different sources of pathogenic bacteria to verify the accuracy of the established detection method. It includes not only low-pathogenic PDD and high-pathogenic PDD strains from different geographical sources, as well as other common pathogens of marine fish, but also introduces another subspecies PDP strain of Photobacterium damselae with high homology, avoiding false positives caused by homologous sequences. In addition, although this experiment only detects the dly gene of a single high-pathogenic PDD, by using 5 high-pathogenic PDD strains from different regions (isolated from the Yellow Sea, Bohai Sea and South China Sea waters in China), it is proved that the detection system has stability for the detection results of PDD strains from different sources. In summary, the two ERA detection methods established in this study have good specificity, and can not only distinguish high-pathogenic PDD from low-pathogenic PDD, but also distinguish Photobacterium damselae subsp. piscicida and other common pathogens of marine fish.

[0107] In order to better clinically apply the detection method established by the present invention, the present invention uses RT-ERA and ERA-LFD to conduct high-pathogenic PDD detection on the samples collected from 132 farms (including 106 tissue samples and 26 water samples), and compares the results with those of basic ERA and conventional PCR. The positive detection rates of RT-ERA, ERA-LFD and basic ERA are the same (positive samples: 30), and are all higher than that of conventional PCR (positive samples: 14), which may be related to the stronger antagonistic effect of ERA on inhibitors in the sample DNA preparation process. The quantitative analysis of RT-ERA shows that when the tissue sample concentration is 400 ng, the copy number of high-pathogenic PDD gene ranges from 1.0 copies to 1.0×10 4 copies; when the water sample concentration is 80 ng, the copy number of this gene in the water sample is lower than 10.0 copies. The detection sample collection areas in our study are widely distributed in the Bohai Sea, Yellow Sea and South China Sea in China. High-pathogenic PDD positives are found in Epinephelus fuscoguttatus♀×E. lanceolatus♂, Epinephelus fuscoguttatus♀×E. polyphekadion♂ and Sebastes schlegelii, and the positive rates in Danzhou (32.1%), Wenchang (38.9% positive rate of Epinephelus fuscoguttatus♀×E. lanceolatus♂), Changdao (28.6% positive rate of Sebastes schlegelii) and Haiyang (25.0% positive rate of Epinephelus fuscoguttatus♀×E. polyphekadion♂) are relatively high.

[0108] We also found through clinical sample testing that highly pathogenic PDD could be detected in the intestinal samples of fish individuals showing significant emaciation symptoms (labeled as T3, T4, T6 - T9, T11) in the F1 farm where Epinephelus intestinal spore disease broke out, with an infection rate as high as 40%. The highly pathogenic PDD may be somewhat correlated with the occurrence of fish emaciation symptoms. There may be a synergistic pathogenic effect between the highly pathogenic PDD and specific aquatic pathogens. This synergistic effect may be related to the destruction of the host tissue barrier mediated by the hemolysin (Dly) secreted by PDD, creating conditions for the invasion of opportunistic pathogens such as Enterospora epinepheli. In addition, highly pathogenic PDD could also be detected in the intestines of fish without obvious clinical symptoms (labeled as T30, T37 - T41, T45 - T47), indicating that the infection of this pathogen is latent. During the latent period, it does not cause obvious symptoms in the host, but once the symptoms appear, the condition may deteriorate rapidly. In Sebastes schlegelii samples, highly pathogenic PDD could be detected in the liver tissue, but not in the intestinal and skin samples. In contrast, highly pathogenic PDD could be detected in the liver, intestine, and skin samples of hybrid grouper, which may be related to the species and the degree of infection.

[0109] We also detected water samples from multiple aquaculture ponds. The detection results of fish and water in the same farm were consistent (F5P1, F7P1, F9P1, and F9P3). Highly pathogenic PDD may be transmitted through water bodies or infected fish. Monitoring the pathogen status in water bodies helps prevent and control diseases. In this study, by detecting the pathogen concentration in water samples, we indirectly evaluated whether fish carried pathogens, thereby predicting the potential severity of the disease, providing an effective means for predicting and monitoring highly pathogenic PDD infection. Through timely detection and early intervention measures (such as treating juvenile fish and replacing water in a timely manner), the horizontal transmission of pathogens can be effectively cut off, preventing disease outbreaks. In addition, this method does not cause harm to fish. By only detecting water samples, the monitoring purpose can be achieved, significantly reducing the economic losses in the aquaculture industry.

[0110] The highly pathogenic PDD RT-ERA and ERA-LFD detection methods established in this invention have the advantages of simple operation, high speed and efficiency, high sensitivity, and strong specificity, and do not require expensive professional equipment. This method can also be used to evaluate the distribution of pathogens in the water environment, providing an important technical basis for the rapid and accurate diagnosis and effective prevention and control of highly pathogenic PDD disease, and at the same time providing strong technical support for the sustainable development of the aquaculture industry.

Claims

1. A method for detecting highly pathogenic Photobacterium mermaniformis subspecies mermaniformis, characterized in that: The Latin name of the highly pathogenic Photobacterium damselae subsp. damselae is Photobacterium damselae subsp. damselae, and it contains a characteristic virulence gene - a damselysin gene sequence; the method comprises using an enzymatic isothermal amplification technology ERA to detect the damselysin gene sequence of the highly pathogenic Photobacterium damselae subsp. damselae; the primer sequence of the ERA is: Pdd-F1; Pdd-R1; from the 5' end to the 3' end, the nucleotide sequence of the Pdd-F1 is sequence number (ID): 1; the nucleotide sequence of the Pdd-R1 is sequence number (ID):

2.

2. The method for detecting the highly pathogenic Photorhabditis mermani subspecies according to claim 1, characterized in that: The length of the primer is 236 bp.

3. The method for detecting the highly pathogenic Photorhabditis mermani subspecies according to claim 1 or 2, characterized in that: The ERA includes real-time fluorescence quantitative combined with ERA or ERA combined with lateral flow chromatography test strips.

4. The method for detecting highly pathogenic Photorhabditis mermani subspecies according to claim 3, characterized in that: The real-time fluorescence quantitative binding ERA probe is Pdd-probe1a. From the 5' end to the 3' end, the nucleotide sequence of Pdd-probe1a is sequence number (ID):

11. A FAM fluorescent group is introduced at the 30th base at the 5' end of sequence number (ID): 11, and a BHQ1 quenching group is designed at the 15th base at the 3' end. A tetrahydrofuran is set between the FAM fluorescent group and the BHQ1 quenching group, and a C3-Spacer is set at the 3' end as a blocking group; The probe of the ERA combined lateral flow chromatography test strip is: Pdd-Sprobe1a. In the direction from the 5' end to the 3' end, the nucleotide sequence of the Pdd-Sprobe1a is sequence number (ID):

12. A FAM fluorescent group is set at the 5' end of sequence number (ID): 12, and a tetrahydrofuran is set at the 30th base of the 5' end, and a C3-Spacer blocking group is set at the 3' end.

5. The method for detecting highly pathogenic Photorhabditis mermani subspecies according to claim 4, characterized in that: The length of the probe is 46 bp.

6. The method for detecting highly pathogenic Photorhabditis mermani subspecies according to claim 3, characterized in that: The primer of the ERA combined lateral flow chromatography test strip is Pdd-R1-biotin, and the Pdd-R1-biotin is labeled by setting a vitamin B7 at the 5' end of Pdd-R1.

7. The method for detecting highly pathogenic Photorhabditis mermani subspecies according to claim 3, characterized in that: The reaction temperature of the real-time fluorescence quantitative binding ERA is 33°C to 41°C, preferably 39°C.

8. The method for detecting highly pathogenic Photorhabditis mermani subspecies according to claim 3, characterized in that: The reaction time of the real-time fluorescence quantitative combined with ERA is 10 to 20 minutes.

9. The method for detecting highly pathogenic Photorhabditis mermani subspecies according to claim 3, characterized in that: The reaction temperature of the ERA combined with lateral flow chromatography test strip is 37° C. to 41° C., preferably 40° C.; the reaction time is 10 to 35 minutes, preferably 10 minutes.

10. The method for detecting highly pathogenic Photorhabditis mermani subspecies according to claim 1 or 2, characterized in that: The detection limit of ERA is 2×10 0 copies / μL.

Images