Method for detecting neonidenia

Abstract

The invention aims to provide a method for detecting neonidenia. The Latin name of the neonidenia litura is Neobenedeniagreslace, and the Latin name of the neonidenia litura is the Neobenedeniagreslace; the method comprises the following steps: detecting a 28S rRNA (ribosomal Ribonucleic Acid) gene sequence of the neonidenia by using an enzymatic isothermal amplification technology (ERA); the primer sequence of the ERA is NEO-PAGE-F4, and the primer sequence of the ERA is The preparation method comprises the following steps: adding an emulsifier, an NEO-HPLC According to the direction from the 5'tail end to the 3 'tail end, the nucleotide sequence of the NEO-PAGE-F4 is a sequence number (ID): 7; the nucleotide sequence of the NEO-HPLC-R2 is shown as a sequence number (ID): 4. The method does not depend on a PCR instrument, has the advantages of being easy and convenient to operate, short in reaction time, high in sensitivity and the like, can detect the pathogenic nucleic acid with the minimum 1 * 10 < 1 > copies / microliter, and greatly improves the detection efficiency.

Description

Technical Field

[0001] The invention relates to a method for detecting Neobenedenia gnathostomia, and belongs to the technical field of detection. Background Art

[0002] Neobenedenia girellae is a monogenetic trematode and one of the main pathogens causing Benedeniasis. Its host range includes many marine farmed fish with high economic value, such as grouper, golden pomfret, large yellow croaker, and various yellowtails. It mainly parasitizes on the head and back of fish. Because the parasite is white and transparent and as big as sesame seeds, the disease is commonly known as "white sesame disease" or "termite disease."

[0003] Benedenia attaches mechanically and absorbs to the host's epidermis through suction cups, destroying skin tissue, causing skin erosion and inflammation, and creating conditions for the invasion of secondary pathogens. The infection not only causes damage to the fish's epidermis, but can also parasitize on mucus cells and blood cells, further aggravating the damage to the fish and significantly reducing the health level and production efficiency of farmed fish. In addition, outbreaks of Benedeniasis are often accompanied by high mortality rates, which directly leads to a decrease in fish production and seriously affects the output value of aquaculture. In order to control the disease, farmers need to invest a lot of manpower and resources in medicated baths or drug feeding, further increasing production costs. The quality of sick fish declines or even the entire group dies, causing significant economic losses to the aquaculture industry.

[0004] Although research on the prevention and control of Benedeniasis has made some progress, there are still many challenges in practical application. Current prevention and control methods mainly focus on post-infection treatment, such as freshwater immersion or drug addition. Although these methods can reduce the number of parasites, their application in large-scale breeding is often limited by complex operations, high costs, and stress on fish. It is difficult to completely remove the parasites and eggs, and it is impossible to fundamentally control the infection. Although the research on chemical drugs and plant extracts provides more options for prevention and control, in actual production, they are still limited by high costs, inconvenient operations, insufficient stability of effects, and difficulty in promotion.

[0005] Therefore, there is still a lack of efficient and widely applicable methods for the prevention and control of Benedeniasis, and early diagnosis and early warning of infection are particularly important. By developing rapid and accurate detection technology, early identification and dynamic monitoring of parasites in water and fish can be achieved, providing a scientific basis for timely and effective prevention and control measures, thereby improving the level of aquaculture health management and reducing disease risks.

[0006] Enzymatic Recombinase Amplification (ERA) is an efficient and sensitive nucleic acid detection technology. This technology achieves nucleic acid amplification under isothermal conditions of 25°C to 45°C by molecularly modifying and optimizing tool enzymes from bacteria, viruses and bacteriophages, without the need for a thermal cycler. ERA technology uses recombinase to assist primers to bind to target sequences, and rapidly extends and amplifies under the action of isothermal polymerase. Compared with traditional PCR, qPCR and loop-mediated isothermal amplification (LAMP), ERA has higher ease of operation, sensitivity and accuracy, and significantly shortens the reaction time, making it particularly suitable for rapid on-site detection. In recent years, ERA technology has gradually been combined with lateral flow test strips (Lateral Flow Device, LFD) to form a technical platform that can detect quickly and visually. By setting the test line and control line on the test strip, the ERA amplification product is combined with the gold-labeled antibody and then colored on the test strip, making the test results intuitive and clear. This type of detection system has the advantages of rapidity, ease of operation and on-site applicability, providing the possibility for rapid diagnosis of a variety of pathogens.

[0007] ERA-LFD technology has been successfully applied to a variety of pathogen detection fields, including the diagnosis of aquaculture pathogens such as Bacillus hepatis and acute hepatopancreatic necrosis disease (AHPND) pathogens in shrimp. For example, the ERA-LFD detection method developed based on the conservative sequences of the pirA and pirB genes of the AHPND pathogen has a sensitivity of 101 copies / μL and has no cross-reaction to other pathogens, providing a rapid on-site diagnostic tool for shrimp farms. In addition, ERA-LFD technology has also been used to detect fish pathogens such as rainbow trout infectious hematopoietic necrosis virus (IHNV), showing significant advantages in shortening diagnosis time and improving detection efficiency. By combining amplification technology with a test strip color development system, this method achieves efficient screening of pathogens without relying on complex instruments and equipment, providing strong support for early warning and prevention and control of aquaculture diseases.

[0008] There is currently no research combining ERA technology with LFD test strips for the detection of the important marine pathogen Neobenedenia. Summary of the invention

[0009] The purpose of the invention is to provide a method for detecting Neobenedenia spp.

[0010] To achieve the purpose of the present invention, the Latin name of the Neobenedenia girellae is Neobenedenia girellae; the method comprises detecting the 28S rRNA gene sequence of the Neobenedenia girellae by using the enzymatic isothermal amplification technology ERA;

[0011] The primer sequences of the ERA are: NEO-PAGE-F4; NEO-HPLC-R2; in the direction from the 5' end to the 3' end, the nucleotide sequence of the NEO-PAGE-F4 is sequence number (ID): 7; the nucleotide sequence of the NEO-HPLC-R2 is sequence number (ID): 4.

[0012] In a specific embodiment, the purification method of the NEO-HPLC-R2 primer is high performance liquid chromatography purification, and the purification method of the NEO-PAGE-F4 primer is polyacrylamide gel electrophoresis.

[0013] In a specific embodiment, the length of the primer is 237 bp.

[0014] In a specific embodiment, the ERA is an ERA combined with a lateral flow chromatography test strip.

[0015] In a specific embodiment, the ERA probe is NEO-HPLC-probe1; in the direction from the 5' end to the 3' end, the nucleotide sequence of the NEO-HPLC-probe1 is sequence number (ID): 13; a FAM fluorescent group is set at the 5' end of sequence number (ID): 13, 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.

[0016] In a specific embodiment, the length of the probe is 47 bp.

[0017] In a specific embodiment, the primer of the ERA-binding lateral flow chromatography test strip is NEO-HPLC-R2-biotin, and the NEO-HPLC-R2-biotin is labeled with vitamin B7 at the 5' end of NEO-HPLC-R2.

[0018] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 39° C. to 46° C., and the reaction time is 10 to 35 min.

[0019] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 43° C. and the reaction time is 10 min.

[0020] In one embodiment, the detection limit of ERA is 1×10 1 copies / μL.

[0021] Beneficial effects:

[0022] 1. The present invention does not rely on PCR instruments and has the characteristics of simple operation, short reaction time, high sensitivity, etc. It can detect pathogenic nucleic acid at a minimum of 1×101 copies / μL, greatly improving the detection efficiency.

[0023] 2. The method of the present invention has good specificity for Neobenedenia gnathostomia and does not cross-react with other common pathogens.

[0024] 3. The method of the present invention can not only evaluate the pathogen load in fish tissues, but also be applied to the detection of pathogens in aquaculture water. Without causing harm to fish, early warning and intervention of pathogens can be achieved through water quality analysis. Once the pathogen is found, the fry can be treated in time and the water quality conditions can be improved, thereby effectively blocking the spread of the pathogen and reducing the economic losses of the farm. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a photo of a greater amberjack infected with Neobenedenia spp. and the worm itself; Figure 1 A, B: The body surface of the amberjack infected with Neobenedenia chussima is covered with white sesame-shaped worm bodies (as indicated by the red arrows); C, D: Photos of Neobenedenia chussima under an optical microscope. The anterior end of the worm body has two parallel anterior haustoria that are like suckers, and the posterior end has an oval posterior haustoria that is much larger than the anterior haustoria.

[0026] Figure 2 The results of primer screening for Neobenedenia sphaeroides, M: D 2000Marker; 1-11: 1-11 represent the 11 primer combinations in Table 1;

[0027] Figure 3 The results of Neobenedenia spp. test in Example 2, A: ERA-LFD test results at different temperatures; B: ERA-LFD test results at different times; NTC: negative control;

[0028] Figure 4 The sensitivity test results of Neobenedenia natans in Example 3; A: ERA-LFD sensitivity test results; B: conventional PCR sensitivity test results; NTC: negative control;

[0029] Figure 5 The specific detection results of Neobenedenia natans in Example 4; A: ERA-LFD specific test results; B: conventional PCR specific results; NTC: negative control;

[0030] Figure 6 The results of conventional PCR testing of 40 samples for Neobenedenia spp. in Example 5;

[0031] A: 1-14 (Table 2 "N1-T14") represents 14 samples of pearl grouper; 15-21 (Table 2 "N15-21") represents 7 samples of golden pomfret; B: 22-40 (Table 3 "W1-W19") represents 19 water samples;

[0032] Figure 7 The results of the basic ERA method in Example 5 for detecting Neobenedenia spp. in 40 samples;

[0033] A: 1-14 (Table 2 "N1-T14") represents 14 samples of pearl grouper; 15-21 (Table 2 "N15-21") represents 7 samples of golden pomfret; B: 22-40 (Table 3 "W1-W19") represents 19 water samples;

[0034] Figure 8 The results of the ERA-LFD method for detecting Neobenedenia spp. in 40 samples in Example 5;

[0035] A: 1~14 (Table 2 "N1~T14") represents 14 samples of pearl grouper; 15~21 (Table 2 "N15-21") represents 7 samples of golden pomfret; B: 22~40 (Table 3 "W1~W19") represents 19 water samples. DETAILED DESCRIPTION

[0036] To achieve the purpose of the present invention, the Latin name of the Neobenedenia girellae is Neobenedenia girellae; the method comprises detecting the 28S rRNA gene sequence of the Neobenedenia girellae by using the enzymatic isothermal amplification technology ERA;

[0037] The primer sequences of the ERA are: NEO-PAGE-F4; NEO-HPLC-R2; in the direction from the 5' end to the 3' end, the nucleotide sequence of the NEO-PAGE-F4 is sequence number (ID): 7; the nucleotide sequence of the NEO-HPLC-R2 is sequence number (ID): 4.

[0038] In a specific embodiment, the purification method of the NEO-HPLC-R2 primer is high performance liquid chromatography purification, and the purification method of the NEO-PAGE-F4 primer is polyacrylamide gel electrophoresis.

[0039] In a specific embodiment, the length of the primer is 237 bp.

[0040] In a specific embodiment, the ERA is an ERA combined with a lateral flow chromatography test strip.

[0041] In a specific embodiment, the ERA probe is NEO-HPLC-probe1; in the direction from the 5' end to the 3' end, the nucleotide sequence of the NEO-HPLC-probe1 is sequence number (ID): 13; a FAM fluorescent group is set at the 5' end of sequence number (ID): 13, 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.

[0042] In a specific embodiment, the length of the probe is 47 bp.

[0043] In a specific embodiment, the primer of the ERA-binding lateral flow chromatography test strip is NEO-HPLC-R2-biotin, and the NEO-HPLC-R2-biotin is labeled with vitamin B7 at the 5' end of NEO-HPLC-R2.

[0044] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 39° C. to 46° C.; and the reaction time is 10 to 35 min.

[0045] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 43° C. and the reaction time is 10 min.

[0046] In one embodiment, the detection limit of ERA is 1×10 1 copies / μL.

[0047] The specific implementation modes of the present invention are further described below in conjunction with embodiments, but the present invention is not limited to the scope of the embodiments.

[0048] 1.1 Main Materials

[0049] Pathogenic materials include Neobenedenia natans, Virbio Parahaemolyticus, Streptococcus agalactiae, Streptococcus iniae, Nervous Necrosis Virus (NNV) cDNA, and Enterospora epinepheli, all of which are tested and preserved in our laboratory.

[0050] The clinical sample experimental materials of this study were obtained from different farms in Hainan Province, including greater amberjack (Seriolarivoliana), leopard gill perch (Plectropomus leopardus), pearl grouper (Epinephelusfuscoguttatus♀×E.lanceolatus♂, hybrid grouper) and golden pomfret (Trachinotus ovatus). Tissue samples of greater amberjack infected with Neobenedenia fuscoguttatus were collected from greater amberjack farms in Hainan Province. Pearl grouper samples were collected in Lingao City, Hainan Province in 2024, including 14 fish tissue samples and 4 cement pond water samples. Golden pomfret samples were collected in Haikou City, Hainan Province, and a total of 7 fish tissue samples and 5 recirculating aquaculture water samples were obtained. In addition, 4 and 6 water samples were collected from cement pond farms in Danzhou City and Dongfang City, Hainan Province, respectively. The collection of these samples provides an important basis for the detection and analysis of Neobenedenia in fish. Some typical symptoms of infected fish are shown in the following pictures. Figure 1 shown.

[0051] 1.2 Main Reagents

[0052] Basic freeze-dried microspheres (ERA method), probe freeze-dried microspheres (ERA method), directional flow test strips (Suzhou Xianda Company, China), FastPure Gel DNA Extraction Mini Kit DC301-01 gel recovery / DNA purification kit (Nanjing Novozyme Biotechnology Co., Ltd., China), tissue genomic DNA extraction kit (Omega Bio-Tek, USA), water genomic DNA extraction kit (Omega Bio-Tek, USA).

[0053] Example 1

[0054] There are 11 primer combinations. In terms of primer purification methods, two methods were selected: polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC). The names of "PAGE" and "HPLC" represent different primer purification methods. The primer combination for conventional PCR is Neob3-F / Neob3-F.

[0055] For the test paper type probe, in the design of the test paper type probe, a FAM fluorescent group is set at the 5' end, THF is added at the 30th base of the 5' end, and a C3-Spacer blocking group is provided at the 3' end. The primer and probe information are shown in Table 1.

[0056] Table 1 Primer and probe information

[0057]

[0058]

[0059] Template DNA extraction

[0060] Total DNA extraction from tissues: DNA of Neobenedenia spp. was extracted according to the instructions of EZNATissue DNAKit D3396 kit of OMEGA Company; about 30 mg of each sample was taken for total tissue DNA extraction, 60 μL of preheated Elution Buffer was added to the center of the adsorption column, and the eluted DNA was stored at -20°C.

[0061] For DNA extraction from water samples, first filter the water sample (3 L) using microporous filter paper (pore size 0.22 μm), cut the filter paper into pieces and put it into a clean 50 mL centrifuge tube, and then extract the total water sample DNA according to the instructions of the EZNA Water DNA Kit D5525-01 kit from OMEGA; use a Nanodrop micro-spectrophotometer to determine the DNA concentration, and store the eluted DNA at -20°C.

[0062] Construction and synthesis of positive plasmid standards

[0063] Using the DNA of Neobenedenia chussima as a template, the primer combination No. 14 in Table 1 was used to amplify the 28SrRNA gene fragment of Neobenedenia chussima. The electrophoresis band position was observed to see whether it was at the expected product length of 357 bp. After confirmation, the amplified fragment containing the target gene was gel purified and recovered using the FastPure Gel DNA Extraction Mini Kit DC301-01 (Nanjing Novogene Biotech Co., Ltd., China).

[0064] The purified product was connected to the PEASY-T1Sample vector CT111 (Beijing Quanshijin Biotechnology Co., Ltd., China) to construct a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells ZC101 (Shanghai Weidi Biotechnology Co., Ltd., China) and spread on LB plates containing ampicillin and cultured in a 37°C constant temperature incubator for 12-16 hours. After a single colony grew, the positive colony was picked and diluted in enzyme-free sterile water. M13 vector primers and colony PCR screening were used. A part of the expected positive clone strains were sequenced and identified, and the other part was inoculated in LB liquid culture medium and cultured overnight. The plasmid was extracted using the Omega Plasmid Mini Kit I plasmid DNA extraction kit D6943 (Omega Bio-Tek, USA). The plasmid DNA concentration was determined to be 168 ng / μL using a Shanghai Instrument spectrophotometer, and the plasmid concentration was calculated to be 3.66×10 based on the Avogadro constant.10 copies / μL, diluted to 1×10 10 The copies / μL were stored at -20°C for later use in ERA detection.

[0065] ERA test for Neobenedenia spp.

[0066] Basic ERA uses basic freeze-dried microspheres (Suzhou Xianda Gene Technology Co., Ltd., China). The basic ERA reaction system consists of 2.1 μL of forward primer (10 μM), 2.1 μL of reverse primer (10 μM), 2 μL of template DNA, and 41.8 μL of enzyme-free water, which form a 48 μL premix. First, add 48 μL of premix to each tube of amplification reagent. Then add 2 μL of ERA activator (containing Mg+) to the test tube cap, quickly centrifuge and mix. First, use each primer combination in Table 1 to perform basic ERA reaction at 45°C and 20 min. After the reaction, add 2.5 μL Protain K to purify the product and run the gel for verification. Select primers with higher brightness and no primer dimer bands for subsequent experiments.

[0067] ERA-LFD detection of Neobenedenia spp.

[0068] Before the experiment, the ERA-LFD kit enzyme freeze-dried powder should be taken out in advance and placed at room temperature for at least 10

[0069] ERA-LFD used probe-type freeze-dried microspheres (ERA method) KS302 and flow test strips TS101 (Suzhou Xianda Gene Technology Co., Ltd., China).

[0070] The primers and probes used for ERA-LFD are shown in Table 1. Add 2.1 μL of forward primer (10 μM), 2.1 μL of reverse primer (10 μM), 0.6 μL of probe (10 μM), and 41.2 μL of enzyme-free water to the tube containing enzyme lyophilized powder to make a 46 μL premix. Then add 2 μL of ERA activator (containing Mg) to the cap of the test tube. + ), add 2μL DNA template to the amplification area, quickly centrifuge and mix, react in a 45℃ constant temperature water bath for 10 minutes, and use a test strip to observe the reaction effect. Use enzyme-free water to dilute the amplification product. The dilution ratio should be 60:1 (that is, take 5μL of reaction product to a 1.5mL centrifuge tube and add 295μL of pure water to dilute), then aspirate 80μL to a new 1.5mL centrifuge tube, and then insert the test strip into the centrifuge tube. Read the results in 7-10 minutes. Under the premise of ensuring that the control line is blue, determine the reaction intensity according to the depth of red of the test line.

[0071] In the ERA primer screening experiment, using 10 ng / μL Neobenedenia sphaeroides DNA as a template, all 11 primer combinations listed in Table 1 successfully amplified the target sequence. On the agarose gel, observe the positive amplification bands, and select primers with higher brightness or no primer dimer bands for subsequent experiments. The experimental results are shown in Figure 1. Figure 2 Primer combination No. 9 (NEO-PAGE-F4 / NEO-PAGE-R2) is the best, and the length of the target product amplified by it is 237 bp.

[0072] In the ERA-LFD experiment, NEO-HPLC-R2 purified by "HPLC" has better resolution and sensitivity than NEO-PAGE-R2 purified by "PAGE", and the test strip experiment can be better visualized. Therefore, the following ERA-LFD method uses the combination of primers and probes listed in Table 1 12.

[0073] Example 2

[0074] ERA-LFD has a 1×10 5 The recombinant plasmid standard with 1000 copies / μL was used as the positive template. The reaction was carried out at 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃ and 46℃ for 10 min. After the reaction, 5μL of the reaction solution was mixed in 295μL of enzyme-free water, and 80μL was aspirated into a new 1.5mL centrifuge tube and tested with a test strip. The experimental results are shown in the figure. Figure 3 As shown in A, the red color of the control band at 43°C is the most obvious, so 43°C is taken as the optimal reaction temperature. With 43°C as the reaction temperature, the reaction was performed for 5min, 10min, 15min, 20min, 25min, 30min and 35min respectively. The results are shown in Figure 3 B. The red color of the control band was very weak at 5 min. During 10 to 35 min, the red color of the control band was very obvious and the color intensity no longer increased. Therefore, 10 min was selected as the optimal reaction time.

[0075] Example 3

[0076] Sensitivity test

[0077] Serial dilutions of the recombinant plasmid standard were performed to adjust the plasmid concentration to 1×10 0 ~1×10 9copies / μL, and the recombinant plasmid standard of Neobenedenia spp. was used as the template for conventional PCR and ERA-LFD detection. The conventional PCR reaction program was: 95℃5min, 94℃30s, 55℃30s, 72℃30s, 72℃5min, repeated 30 cycles. The reaction system was: Taq MasterMix 12.5μL, ddH2O 9.5μL, forward primer 1μL, reverse primer 1μL, DNA template 1μL. The reaction system for ERA-LFD sensitivity detection was: forward primer 2.1μL (10μM), reverse primer 2.1μL (10μM), template DNA 2μL, probe 0.6μL, enzyme-free water 41.2μL, activator 2μL. Reaction conditions: 43℃, 10min.

[0078] ERA-LFD test results are as follows Figure 4 As shown in A, at a concentration of 1×10 1 ~1×10 9 The test strips in the test line of the copies / μL range all showed red bands, so the minimum detection limit of the ERA-LFD method established in this study was 1×10 1 copies / μL. Conventional PCR test results are as follows Figure 4 As shown in B, at a concentration of 1×10 9 ~1×10 4 The bands were shown in the range of copies / μL, and the lowest detection limit was 1×10 4 copies / μL.

[0079] Example 4

[0080] ERA-LFD and conventional PCR were performed using Neobenedenia chusii, Vibrio parahaemolyticus, Streptococcus agalactiae, Streptococcus dolphinus, cDNA of grouper nervous necrosis virus, and Enterospora spp. as templates. The established ERA-LFD reaction method and conventional PCR were used to detect Neobenedenia chusii, Vibrio parahaemolyticus, Streptococcus agalactiae, Streptococcus dolphinus, cDNA of grouper nervous necrosis virus, and Enterospora spp. The conventional PCR reaction program was: 95℃5min, 94℃30s, 55℃30s, 72℃30s, 72℃5min, repeated for 30 cycles. The reaction system was: Taq Master Mix 12.5μL, ddH2O 9.5μL, forward primer 1μL, reverse primer 1μL, DNA template 1μL. The reaction system of ERA-LFD is: forward primer 2.1μL (10μM), reverse primer 2.1μL (10μM), template DNA 2μL, probe 0.6μL, enzyme-free water 41.2μL, activator 2μL. Reaction conditions: 43℃, 10min. The results are as follows Figure 5 A and Figure 5 As shown in B, the test strip detection line and gel running results using only the DNA sample of Neobenedenia gnatogensis as the template showed red bands and amplified bands, respectively, while other pathogens and negative controls were all negative, indicating that the two established methods have good specificity for Neobenedenia gnatogensis.

[0081] Example 5

[0082] Clinical tissue and water sample testing

[0083] ERA-LFD was used to detect Neobenedenia spp. in 21 tissue samples (Table 2) and 19 water samples (Table 3) collected in 2024, and the results were compared with those of conventional PCR and basic ERA. The reaction system of ERA-LFD was: forward primer 2.1 μL (10 μM), reverse primer 2.1 μL (10 μM), template DNA 2 μL, probe 0.6 μL, enzyme-free water 41.2 μL, activator 2 μL. The conventional PCR reaction program was: 95℃5min, 94℃30s, 55℃30s, 72℃30s, 72℃5min, repeated 30 cycles. The reaction system was: Taq Master Mix 12.5 μL, ddH2O 9.5 μL, forward primer 1 μL, reverse primer 1 μL, DNA template 1 μL. The reaction system of basic ERA is 2.1μL (10μM) of forward primer, 2.1μL (10μM) of reverse primer, 2μL of template DNA, 41.8μL of enzyme-free water, and 2μL of activator. The positive results of conventional PCR, basic ERA, and ERA-LFD were counted (Table 4). Specific gel images of each detection method: Among them, the conventional PCR method detected 22 positive samples: 6 samples in Lingao City, 6 tissue samples in Haikou City, and 10 water samples. The results are shown in Figure 6 The basic ERA and ERA-LFD methods detected 26 positive samples: 10 tissue samples from Lingao City, 6 tissue samples from Haikou City, and 10 water samples. Figure 7 , Figure 8 The positive rate of tissue and water samples was consistent with that of ERA-LFD, with a positive rate of 76.2% for Neobenedenia spp. in tissue samples and 52.6% in water samples. The positive rates of conventional PCR in tissue and water samples were 57.1% and 52.6%, respectively.

[0084] Table 2. Detection results of conventional PCR, basic ERA and ERA-LFD on clinical fish tissue samples

[0085]

[0086] Note: - represents the test result of Neobenedenia chussima is negative; + represents the test result of Neobenedenia chussima is positive.

[0087] Table 3. Detection results of conventional PCR, basic ERA and ERA-LFD on environmental water samples

[0088]

[0089] Note: - represents the test result of Neobenedenia chussima is negative; + represents the test result of Neobenedenia chussima is positive.

[0090] Table 4 Statistics of the detection results of Neobenedenia spp. by basic ERA, ERA-LFD and conventional PCR

[0091]

[0092] Neobenedenia girellae is one of the most serious monogenetic trematodes in aquaculture and fisheries worldwide. It infects a large number of economically valuable fish, causing epithelial tissue degeneration, dermal erosion and inflammatory response through mechanical attachment and gnawing on the host epidermis, and further triggering secondary pathogen infection. The present invention successfully established a rapid detection method based on ERA-LFD for this important pathogen, providing an efficient tool for early diagnosis and prevention and control of Neobenedenia girellae.

[0093] ERA technology does not require complex instruments such as PCR instruments, and can be completed through constant temperature amplification. It has the characteristics of simple operation, short reaction time and high sensitivity. The present invention finally established the NEO-PAGE-F4 / NEO-HPLC-R2 primer pair and the NEO-HPLC-probe1 probe. Within 10 minutes of constant temperature reaction at 43°C, the results can be observed by the ERA-LFD detection method, and the detection sensitivity is as high as 1×10 1 The ERA-LFD method has good specificity for Neobenedenia spp. and does not cross-react with other common pathogens.

[0094] In actual sample testing, the ERA-LFD method showed a high positive detection rate. The test results of 40 actual samples showed that the positive detection rate of ERA-LFD was the same as that of the basic ERA method (76.2% for tissue samples and 52.6% for water samples), which was significantly higher than the positive detection rate of conventional PCR (57.1% for tissue samples and 52.6% for water samples). This further verifies the high sensitivity and reliability of the ERA-LFD method, and shows that it is suitable for rapid diagnosis needs in actual production.

[0095] It is worth mentioning that the present invention not only realizes the detection of Neobenedenia in tissue samples, but also uses ERA-LFD technology to analyze the pathogen concentration in water samples. By detecting pathogens in water bodies, the pathogen carrying situation of fish and the severity of diseases can be indirectly evaluated, thereby providing a comprehensive disease monitoring method for farms. This method has the advantage of non-destructive testing, and can achieve early warning and intervention of pathogens through water quality analysis without causing harm to fish. Once the pathogen is found, the fry can be treated and the water quality conditions can be improved in time, thereby effectively blocking the spread of pathogens and reducing the economic losses of farms.

[0096] In summary, the ERA-LFD detection method established in the present invention not only overcomes the dependence of conventional nucleic acid detection methods on complex instruments and long-term reactions, but also shows obvious advantages in sensitivity and practicality, providing efficient and reliable technical support for the prevention and control of Neobenedenia in fishery production. The promotion and application of this method will help reduce the economic losses caused by aquaculture diseases and promote the healthy and sustainable development of the aquaculture industry.

Claims

1. A method for detecting Neobenedenia natans, characterized in that: The Latin name of the Neobenedenia girellae is Neobenedenia girellae; the method comprises detecting the 28SrRNA gene sequence of the Neobenedenia girellae by using the enzymatic isothermal amplification technology ERA; The primer sequences of the ERA are: NEO-PAGE-F4; NEO-HPLC-R2; in the direction from the 5' end to the 3' end, the nucleotide sequence of the NEO-PAGE-F4 is sequence number (ID): 7; the nucleotide sequence of the NEO-HPLC-R2 is sequence number (ID):

4.

2. The method for detecting Neobenedenia natans according to claim 1, wherein The purification method of the NEO-HPLC-R2 primer is high performance liquid chromatography purification, and the purification method of the NEO-PAGE-F4 primer is polyacrylamide gel electrophoresis.

3. The method for detecting Neobenedenia natans according to claim 1 or 2, characterized in that, The length of the primer is 237 bp.

4. The method for detecting Neobenedenia natans according to claim 1 or 2, characterized in that, The ERA is an ERA combined with a lateral flow chromatography test strip.

5. The method for detecting Neobenedenia natans according to claim 4, wherein: The ERA probe is NEO-HPLC-probe1; in the direction from the 5' end to the 3' end, the nucleotide sequence of the NEO-HPLC-probe1 is sequence number (ID): 13; a FAM fluorescent group is set at the 5' end of sequence number (ID): 13, 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.

6. The method for detecting Neobenedenia natans according to claim 5, characterized in that: The length of the probe is 47 bp.

7. The method for detecting Neobenedenia natans according to claim 4, characterized in that: The primer of the ERA combined lateral flow chromatography test strip is NEO-HPLC-R2-biotin, and the NEO-HPLC-R2-biotin is labeled by setting a vitamin B7 at the 5' end of NEO-HPLC-R2.

8. The method for detecting Neobenedenia natans according to claim 4, characterized in that: The reaction temperature of the ERA combined with the lateral flow chromatography test strip is 39° C. to 46° C., and the reaction time is 10 to 35 minutes.

9. The method for detecting Neobenedenia natans according to claim 8, characterized in that: The reaction temperature of the ERA combined with the lateral flow chromatography test strip is 43° C. and the reaction time is 10 min.

10. The method for detecting Neobenedenia natans according to claim 1 or 2, characterized in that: The detection limit of ERA is 1×10 1 copies / μL.

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