Detection method of grouper enterosporidia

Abstract

The invention aims to provide a method for detecting enterosporidia of groupers. The Latin name of the enterosporidium of the grouper is Enterospora epinepheli, and the Latin name of the enterosporidium of the grouper is Enterospora epinepheli; the method comprises the following steps: detecting the 18SrRNA gene sequence of the enterosporidia of the grouper by using an enzymatic isothermal amplification technology (ERA); the primer sequence of the ERA is EEP-HPLC (High Performance Liquid Chromatography)-F2; and EEP-HPLC-R2 (Ethylene Propylene Phosphate According to the direction from the 5'tail end to the 3 'tail end, the nucleotide sequence of the EEP-HPLC-F2 is a sequence number (ID): 25; and the nucleotide sequence of the EEP-HPLC-R2 is as shown in a sequence number (ID): 26. The method does not depend on professional PCR instruments and constant-temperature operation, rapid detection and high sensitivity show huge potential in aquatic pathogen detection, and compared with conventional PCR and other nucleic acid amplification methods, the method has the advantages of being easy to operate, efficient, high in sensitivity and high in practicability.

Description

Technical Field

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

[0002] Grouper (Epinephelus spp.) is a valuable and economically valuable marine farmed fish, with a farmed volume of more than 200,000 tons in 2022. However, in recent years, its aquaculture industry has been seriously threatened by an intestinal microsporidia (Latin name Enterospora epinepheli), especially in the seedling stage. The pathogen was first discovered in intestinal samples of juvenile grouper with emaciation symptoms in a hatchery in 2016. It mainly parasitizes in the host intestine, causing symptoms such as anorexia, emaciation, thinning of the intestinal wall and white stool in the fish, and its pathological effects can spread to the liver and other organs. The pathogen is prevalent in the main grouper farming areas of Fujian, Guangdong, Hainan and Guangxi, and can be horizontally transmitted between fish bodies, with a mortality rate of up to 100%. The degree of harm is second only to grouper neuronecrosis virus disease, causing serious economic losses to the grouper farming industry. Because it parasitizes in the host intestinal epithelial cells and has a high degree of concealment, there is currently no effective prevention and control method. Therefore, regular monitoring and early accurate diagnosis are crucial for the prevention and control of diseases caused by this pathogen.

[0003] At present, the detection technologies of E. coli in grouper include optical microscopy (OM), electron microscopy (EM), polymerase chain reaction (PCR) and quantitative PCR (TaqMan qPCR) based on TaqMan probe method. Although OM and EM are of great value in the study of pathogen morphology, they have limitations such as long observation time, slow reaction speed, complex instruments and equipment, cumbersome operation steps, and insufficient sensitivity and specificity. Although TaqMan qPCR has advantages in sensitivity, its high requirements for professional experimental equipment and environment limit its application in rapid detection and resource-constrained environments, especially when early diagnosis of occult diseases is required. Faced with the limitations of existing detection technologies, this study is committed to developing an efficient, sensitive and easy-to-operate detection technology, aiming to provide a more effective solution for the early diagnosis and rapid screening of E. coli in grouper.

[0004] Enzymatic recombinase amplification (ERA) is a constant temperature nucleic acid amplification technology. It has been successfully applied in aquatic pathogen detection due to its simplicity, rapidity and high efficiency. It breaks through the dependence of traditional PCR on large instruments and realizes the rapid amplification of pathogen nucleic acids under a wide range of constant temperature conditions from 25 to 42°C. The amplification efficiency can reach billions of times in 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) has the advantage of on-site detection without the need for precise temperature control equipment with its rapid visualization of detection results within 10 minutes. There has been no report on the use of ERA in the detection of grouper enterosporidia. Summary of the invention

[0005] The invention aims to provide a method for detecting grouper enterosporidia.

[0006] To achieve the purpose of the present invention, the Latin name of the grouper enterospora is Enterospora epinepheli; the method comprises detecting the 18S rRNA gene sequence of the grouper enterospora by using the enzymatic isothermal amplification technology ERA;

[0007] The primer sequences of the ERA are: EEP-HPLC-F2; EEP-HPLC-R2; in the direction from the 5' end to the 3' end, the nucleotide sequence of the EEP-HPLC-F2 is sequence number (ID): 25; the nucleotide sequence of the EEP-HPLC-R2 is sequence number (ID): 26.

[0008] In a specific embodiment, the primer is purified by high performance liquid chromatography.

[0009] In a specific embodiment, the length of the primer is 206 bp.

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

[0011] In a specific embodiment, the real-time fluorescence quantitative probe combined with ERA is:

[0012] EEP-probe2a; in the direction from the 5' end to the 3' end, the nucleotide sequence of the EEP-probe2a is sequence number (ID): 49; a FAM-dT fluorescent group is set at the 30th base of the 5' end of sequence number (ID): 49, and a BHQ1-dT quenching group is set at the 15th base of the 3' end, a C3-Spacer blocking group is set at the 3' end, and a tetrahydrofuran is set between the fluorescent group and the quenching group;

[0013] The probe of the ERA combined lateral flow chromatography test strip is: EEP-Sprobe2a, and the nucleotide sequence of the EEP-Sprobe2a is sequence number (ID): 50; a FAM fluorescent group is set at the 5' end of sequence number (ID): 50, 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.

[0014] In a specific embodiment, the length of the probe is 46 bp.

[0015] In a specific embodiment, the primer of the ERA-binding lateral flow chromatography test strip is EEP-R2-biotin, and the EEP-R2-biotin is labeled with vitamin B7 at the 5' end of EEP-HPLC-R2.

[0016] In a specific embodiment, the reaction temperature of the real-time fluorescence quantitative binding ERA is 33° C. to 41° C., preferably 39° C.; the reaction time is preferably 14 to 20 min.

[0017] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 37°C to 40°C, preferably 40°C; the reaction time is 5 to 35 min, preferably 10 min.

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

[0019] Beneficial effects:

[0020] The method of the present invention does not rely on professional PCR instruments and constant temperature operation, and has rapid detection and high sensitivity, showing great potential in aquatic pathogen detection. Compared with conventional PCR and other nucleic acid amplification methods, it has the advantages of simple operation, high efficiency and high sensitivity, and is more practical.

[0021] The ERA method of the present invention can overcome the influence of inhibitors in the sample DNA preparation process and improve the sensitivity of detection.

[0022] The RT-ERA of the present invention is combined with a portable fluorescence quantitative instrument, and can accurately judge the results through the quantitative curve within a time range of about 33°C to 41°C and 14 to 20 minutes. The results of ERA-LFD are clearly interpreted, and the results can be obtained within 5 to 35 minutes at 37°C to 40°C, which is clearer and more intuitive than the basic ERA and PCR through the band interpretation on the gel map.

[0023] The method of the present invention uses plasmid standard as template, and the detection limit of both methods is 2×10 0 The detection rate was 2.3449 copies / μL, with good specificity, and no cross-reaction with common pathogens of grouper such as amylocyanine dinoflagellates, Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Photobacterium dasyphylum subspecies dasyphyllum (PDD), Streptococcus dolphins, grouper neural necrosis virus (VNN) and grouper iridovirus (SGIV).

[0024] The method of the present invention can not only evaluate the pathogen load in fish tissues, but also can be applied to the detection of pathogens in aquaculture water, and has important value for the early warning and prevention and control of grouper enterosporidiosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The infection status of Enterospora in Pearl Grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂);

[0026] A: A slightly emaciated grouper (top) and a severely emaciated grouper (bottom); B: The intestinal wall of the grouper infected with Enteric cysts becomes thinner and the contents decrease; C: The intestines of the diseased grouper (a, b, c, d): the intestinal wall becomes thinner and transparent, and white contents remain in the intestine, of which a and b excrete white feces; The intestines of a slightly emaciated grouper (e, f); Red arrows: sunken abdominal cavity; Blue arrows: flattened intestines.

[0027] Figure 2 Example 1 Detection results of target sequences amplified by different primer probes.

[0028] Figure 3 Test results of Examples 2-4; NTC: negative control.

[0029] Figure 4 Example 5 Sensitivity test results; NTC: negative control.

[0030] Figure 5 Example 6 Specific detection results of Enterospora in grouper; NTC: negative control.

[0031] Figure 6The conventional PCR method of Example 7 is used to detect the intestinal spores of grouper of 68 samples collected from Hainan grouper farms; among them, 1-28, 30-34, 38-40 (Table 2 "T1-T28, T30-T34, T38-T40") represent 36 pearl grouper; 29, 35-37, 41, 42 (Table 2 "T29, T35-T37, T41, T42") represent 6 leopard gill perch samples, and 43-68 (Table 3 "W1-W26") represent 26 water samples.

[0032] Figure 7 The results of the detection of enterospora in grouper of 68 samples collected from Hainan grouper farms by the basic ERA method of Example 7; among them, 1-28, 30-34, 38-40 (Table 2 "T1-T28, T30-T34, T38-T40") represent 36 samples of pearl grouper; 29, 35-37, 41, 42 (Table 2 "T29, T35-T37, T41, T42") represent 6 samples of leopard gill perch, and 43-68 (Table 3 "W1-W26") represent 26 water samples.

[0033] Figure 8 The RT-ERA test result of the copy number of Epinephelus ocellaris in the positive sample of Example 7;

[0034] A: Applicability of the RT-ERA detection method. RT-ERA detection results of Epinephelus grouper in 68 samples collected from Hainan grouper farms; B: Calculation of the copy number of Epinephelus gene in 29 positive samples out of 68 samples based on Ct values.

[0035] Fig. 9 The ERA-LFD method of Example 7 is used to detect the intestinal spores of 68 samples of grouper collected from the Hainan grouper farm; among them, 1-28, 30-34, 38-40 (Table 2 "T1-T28, T30-T34, T38-T40") represent 36 samples of pearl grouper; 29, 35-37, 41, 42 (Table 2 "T29, T35-T37, T41, T42") represent 6 samples of leopard gill perch, and 43-68 (Table 3 "W1-W26") represent 26 water samples. DETAILED DESCRIPTION

[0036] To achieve the purpose of the present invention, the Latin name of the grouper enterospora is Enterosporaepinepheli; the method comprises detecting the 18S rRNA gene sequence of the grouper enterospora by using the enzymatic isothermal amplification technology ERA;

[0037] The primer sequences of the ERA are: EEP-HPLC-F2; EEP-HPLC-R2; in the direction from the 5' end to the 3' end, the nucleotide sequence of the EEP-HPLC-F2 is sequence number (ID): 25; the nucleotide sequence of the EEP-HPLC-R2 is sequence number (ID): 26.

[0038] In a specific embodiment, the primer is purified by high performance liquid chromatography.

[0039] In a specific embodiment, the length of the primer is 206 bp.

[0040] In a specific embodiment, the ERA includes real-time fluorescence quantitative combined with ERA or ERA combined with lateral flow chromatography test strips.

[0041] In a specific embodiment, the real-time fluorescence quantitative probe combined with ERA is:

[0042] EEP-probe2a; in the direction from the 5' end to the 3' end, the nucleotide sequence of the EEP-probe2a is sequence number (ID): 49; a FAM-dT fluorescent group is set at the 30th base of the 5' end of sequence number (ID): 49, and a BHQ1-dT quenching group is set at the 15th base of the 3' end, a C3-Spacer blocking group is set at the 3' end, and a tetrahydrofuran is set between the fluorescent group and the quenching group;

[0043] The probe of the ERA combined lateral flow chromatography test strip is: EEP-Sprobe2a, and the nucleotide sequence of the EEP-Sprobe2a is sequence number (ID): 50; a FAM fluorescent group is set at the 5' end of sequence number (ID): 50, 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.

[0044] In a specific embodiment, the length of the probe is 46 bp.

[0045] In a specific embodiment, the primer of the ERA-binding lateral flow chromatography test strip is EEP-R2-biotin, and the EEP-R2-biotin is labeled with vitamin B7 at the 5' end of EEP-HPLC-R2.

[0046] In a specific embodiment, the reaction temperature of the real-time fluorescence quantitative binding ERA is 33° C. to 41° C., preferably 39° C.; the reaction time is preferably 14 to 20 min.

[0047] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 37°C to 40°C, preferably 40°C; the reaction time is 5 to 35 min, preferably 10 min.

[0048] In one embodiment, the detection limit of ERA is 2×10 0 copies / μL.

[0049] 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.

[0050] 1.1 Main Materials

[0051] DNA of grouper Enterospora-positive tissues, DNA of Amyloodinium ocellatum, DNA of Cryptocaryon irritans, DNA of Vibrio alginolyticus, DNA of V. harveyi, DNA of V. Parahaemolyticus, DNA of Photobacterium damselae subsp. damselae (PDD), DNA of Streptococcus iniae, DNA of Singapore grouper iridovirus (SGIV) and cDNA of viral nervous necrosis (VNN) of grouper were all detected and preserved by our laboratory.

[0052] In 2023, 28 tissue samples of Epinephelus fuscoguttatus♀×E.lanceolatus♂, Hybrid grouper were collected from a farm in Wenchang City, Hainan Province, China, where an outbreak of Epinephelus fuscoguttatus infection occurred. In 2024, 14 tissue samples of Epinephelus fuscoguttatus and Plectropomus leopardus and 26 water samples from cement ponds were collected from Chengmai County, Danzhou City and Lingao County, Hainan Province. Some photos of fish with typical symptoms are shown below. Figure 1 shown.

[0053] 1.2 Main Reagents

[0054] ERA basic nucleic acid amplification kit, ERA fluorescent nucleic acid amplification kit, ERA test paper nucleic acid amplification kit, flow test strips (Suzhou Xianda Company, China), tissue genomic DNA extraction kit (Omega Bio-Tek Company, USA), water genomic DNA extraction kit (Omega Bio-Tek Company, USA), Super Total RNA Extraction Kit (LS1040) total RNA extraction kit (Promega (Beijing) Biotechnology Co., Ltd., China), Fast Pure Gel DNA Extraction Mini Kit DC301-01 gel recovery / DNA purification kit (Nanjing Novogene Biotech Co., Ltd., China).

[0055] Example 1

[0056] There are 24 primer combinations. For primer purification, two methods, polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC), were selected. The names with "PAGE" and "HPLC" represent different primer purification methods. Combination 1 and combination 12, combination 2 and combination 13, combination 3 and combination 14, combination 4 and combination 15, combination 5 and combination 16, combination 6 and combination 19, combination 7 and combination 20, combination 8 and combination 21, combination 9 and combination 22, combination 10 and combination 23, combination 11 and combination 24 adopted PAGE and HPLC purification methods respectively.

[0057] In the design of the fluorescent probe, we introduced a FAM fluorescent group at the 30th base at the 5' end and designed a BHQ1 quencher group at the 15th base at the 3' end. To ensure the effective transmission of the fluorescent signal, we used an abasic nucleotide analog (tetrahydrofuran, THF) between the fluorescent group and the quencher group as a spacer, and set a C3-Spacer as a blocking group at the 3' end. For the test paper type probe, we also set a FAM fluorescent group at the 5' end, added THF at the 30th base at the 5' end, and equipped the 3' end with a C3-Spacer blocking group. This design ensures that the total length of both probes is 46 base pairs, optimizing the detection performance. The primer combination for conventional PCR is EEP-PAGE-F2 / EEP-PAGE-R2. Primer and probe information is shown in Table 1.

[0058] Table 1 Primer and probe information

[0059]

[0060]

[0061] Construction and synthesis of positive plasmid standards

[0062] Using the positive intestinal tissue samples infected with E. grouper as templates, the conserved sequences in the 18S rRNA gene of E. grouper were amplified using the EEP-HPLC-F2 / EEP-HPLC-R2 primer combination (Table 1). The amplified fragment was gel purified and recovered using the FastPureGel DNA Extraction Mini Kit DC301-01 (Nanjing Novogene Biotech Co., Ltd., China), and the purified product was ligated into the PEASY-T1Sample vector CT111 (Beijing Quanshijin Biotech Co., Ltd., China) to construct the recombinant plasmid EEP-XL-2018a. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells ZC101 (Beijing Zhuangmeng International Biogene Technology Co., Ltd., China), and spread on LB plates containing ampicillin and cultured in a constant temperature incubator at 37°C until colonies grew. Positive colonies were picked and diluted in enzyme-free sterile water, screened using M13 vector primers and colony PCR, and sequenced for identification. After the positive clone strain was inoculated into LB liquid medium and cultured overnight, the plasmid was extracted using Omega Plasmid Mini Kit I Plasmid DNA Extraction Kit D6943 (Omega Bio-Tek, USA). The plasmid DNA concentration was determined to be 123 ng / μL by Shanghai Instruments spectrophotometer, and the plasmid concentration was calculated to be 2×10 10 copies / μL, stored at -20°C for later use in ERA detection.

[0063] Template DNA extraction

[0064] Total DNA extraction from tissues: DNA from the midgut tissue (including intestinal contents) of grouper was extracted according to the instructions of the EZNATissue DNAKit D3396 kit of OMEGA Company; about 30 mg of each sample was taken for total tissue DNA extraction, and then the DNA concentration was determined using a Nanodrop micro-spectrophotometer and uniformly controlled to 200 ng / μL (if the concentration was too low, re-extract it; if the concentration was too high, dilute it with the Elutionbuffer in the kit), and the eluted DNA was stored at -20°C.

[0065] For DNA extraction from water samples, first filter the water sample (5 L) using microporous filter paper (pore size 0.22 μm), cut the filter paper into four pieces and put them 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 uniformly control it to 50 ng / μL (if the concentration is too low, re-extract it; if the concentration is too high, dilute it with the Elution buffer in the kit), and store the eluted DNA at -20°C.

[0066] Basic ERA test

[0067] Basic ERA was performed using the GenDx ERA Basic Nucleic Acid Amplification Kit KS101 (Suzhou XinDa Gene Technology Co., Ltd., China).

[0068] The basic ERA reaction system 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 solvent, and 21 μL of enzyme-free water, which together 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 cap of the test tube. + ), centrifuge quickly and mix. First, use the 24 different primer combinations in Table 1 to perform a basic ERA reaction test at 39°C for 15 minutes.

[0069] RT-ERA assay

[0070] RT-ERA was performed using the GenDx ERA Fluorescence Nucleic Acid Amplification Kit KS103 (Suzhou Xinda Gene Technology Co., Ltd., China).

[0071] The RT-ERA reaction system consists of 48 μL of premixed solution consisting 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 dissolving agent, and 21.2 μL of enzyme-free water. First, add 48 μL of premixed solution to each tube of amplification reagent. Then add 2 μL of ERA activator (containing Mg) to the test tube cap. +), centrifuge quickly and mix. Use Q2000B real-time fluorescence quantitative PCR instrument (Hangzhou Langji Technology Instrument Co., Ltd., China) to detect the fluorescence value. The experimental type is set to absolute quantification, the dye is set to TaqMan Reagents, the reaction program is set to 40 cycles and the fluorescence value is read every 30 seconds. The amplification curve of the positive template is "S" shaped, while the negative control has no amplification. The point where the set threshold (300CFU) intersects with the amplification curve is the Ct value. The degree of inclination 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. The primers and probes are shown in Table 1 above, the reaction temperature used is 39°C, and the reaction time is 20min.

[0072] ERA-LFD detection

[0073] ERA-LFD used the GenDx ERA test paper-based nucleic acid amplification kit KS105 and flow test strips TS101 (Suzhou Xinda Gene Technology Co., Ltd., China).

[0074] 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, 20 μL of solvent, and 21.2 μL of enzyme-free water, which together 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. + ), centrifuge quickly and mix. When using the test strip to observe the reaction effect, the amplification product needs to be diluted with enzyme-free water, and the dilution ratio should be 40:1 (that is, take 5μL of the reaction product to a 1.5mL centrifuge tube and add 200μL of pure water to dilute), then insert the test strip into the centrifuge tube, and 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 the red color of the test line. The primers and probes are shown in Table 1 above, the reaction temperature used is 40℃, and the reaction time is 20min.

[0075] 2×10 5 The recombinant plasmid standard with 100 copies / μL was the positive template, and all primer combinations listed in Table 1 successfully amplified the target sequence. M: D 2000Marker, 1-24: 1-24 primer combinations. On the agarose gel, primer combinations 1, 2, 4, 6-9, 11, 14, 15, 17-22, 24 produced non-specific bands; the bands of primer combinations 3, 5, and 16 were lighter in color; the bands of primer combinations 10, 12, and 23 were smaller in size. The experimental results are shown in Figure 1. Figure 2As shown in the figure, primer combination 13 (EEP-HPLC-F2 / EEP-HPLC-R2) can amplify a target product of 206 bp in length. In addition, primer purification using HPLC will produce brighter bands than PAGE.

[0076] In the following RT-ERA and ERA-LFD experiments, the primer and probe combination used in the RT-ERA method was EEP-HPLC-F2 / EEP-HPLC-R2 / EEP-probe2a probe. The primer and probe combination used in the ERA-LFD method was EEP-HPLC-F2 / EEP-R2-biotin / EEP-Sprobe2a probe.

[0077] Example 2

[0078] RT-ERA assay, 2 × 10 5 The recombinant plasmid standard with 1000 copies / μL was used as the positive template. The real-time fluorescence quantitative ERA was set to 40 cycles, each cycle lasting 30 seconds, and the reaction was carried out at 33°C, 35°C, 37°C, 39°C, and 41°C for 20 minutes. The experimental results are shown in Figure 2. Figure 3 As shown in Figure A, the amplification curve at 39°C has the smallest Ct value, the largest slope and the highest fluorescence intensity. The reaction reaches a plateau within 28 to 40 cycles, so a reaction time of 14 to 20 minutes is optimal.

[0079] Example 3

[0080] ERA-LFD detection, with 2×10 5 The recombinant plasmid standard with 1000 copies / μL was used as the positive template, and the reaction was carried out at 37℃, 38℃, 39℃, and 40℃ for 20 minutes. After the reaction, 5μL of the reaction solution was mixed in 200μL of enzyme-free water and tested with a test strip. Figure 3 As shown in Figure B, the red color of the control band at 40°C is the most obvious.

[0081] Example 4

[0082] ERA-LFD detection: The reaction temperature was 40°C, and the reaction time was 5 min, 10 min, 15 min, 20 min, 25 min, 30 min and 35 min respectively. The results were as follows: Figure 3 As shown in Figure C, the red color of the control band is very weak at 5 minutes, and is very obvious during 10 to 35 minutes, and the color depth does not deepen with time. The best color development time is 10 minutes.

[0083] Example 5

[0084] Sensitivity test

[0085] At a concentration of 2×10 0 ~2×10 8 The recombinant plasmid standard with 100 copies / μL was used as a template for conventional PCR, basic ERA, RT-ERA and ERA-LFD detection. Conventional PCR reaction system: The primers used were EEP-PAGE-F2 / EEP-PAGE-R2 (Table 1), and the reaction system (50 μL) contained 2 μL forward primer (concentration 10 μmol / L), 2 μL reverse primer (concentration 10 μmol / L), 2 μL template DNA, 25 μL 2×Rapid Taq Master Mix and dd water to 50 μL. Conventional PCR reaction conditions DNA denaturation was performed at 95°C for 5 minutes, followed by a total of 35 PCR cycles, with the following specific conditions: DNA denaturation at 94°C for 30 seconds, primer annealing at 57°C for 30 seconds, and DNA polymerization at 72°C for 1 minute. After the last cycle, the reaction was terminated at 72°C for 10 minutes. Basic ERA: EEP-HPLC-F2 / EEP-HPLC-R2 (Table 1), reaction temperature and time: 39℃ for 15min. RT-ERA: EEP-HPLC-F2 / EEP-HPLC-R2 / EEP-probe2a (Table 1), reaction temperature and time: 39℃ for 20min. ERA-LFD: EEP-HPLC-F2 / EEP-HPLC-R2 / EEP-Sprobe2a (Table 1), reaction temperature and time: 40℃ for 10min. The results of basic ERA are as follows: Figure 4 As shown in Figure A, at a concentration of 2×10 0 ~2×10 8 The bands were shown in the range of copies / μL, and the lowest detection limit was 2×10 0 copies / μL. ERA-LFD test results are as follows Figure 4 As shown in Figure B, at a concentration of 2×10 0 ~2×10 8 The test strips in the test line of the copies / μL range all showed red bands, indicating that the detection limit of the ERA-LFD method of the present invention is 2×10 0 copies / μL. RT-ERA test results are as follows Figure 4 As shown in Figure C, amplification curves appeared in the three biological replicates of each concentration gradient, indicating that the detection limit of the RT-ERA method of the present invention is 2×10 0 The standard curve was drawn based on the relationship between the recombinant plasmid copy number (x) and the threshold cycle number Ct value (y), and the formula was y = -2.1226x + 19.562 (e.g. Figure 4 (as shown in Figure D), the correlation coefficient R2 =0.9915.

[0086] Example 6

[0087] The RT-ERA and ERA-LFD reaction methods were used to detect the starch egg dinoflagellate, Cryptocaryon irritans, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Photobacterium mermaidense, Streptococcus dolphinus, grouper nervous necrosis virus and grouper iris virus. RT-ERA: EEP-HPLC-F2 / EEP-HPLC-R2 / EEP-probe2a (Table 1), reaction temperature and time: 39℃ for 16min. ERA-LFD: EEP-HPLC-F2 / EEP-HPLC-R2 / EEP-Sprobe2a (Table 1), reaction temperature and time: 40℃ for 10min. The results are as follows. Figure 5 China Library A and Figure 5 As shown in Figure B, only the real-time fluorescence quantitative reaction and test strip detection line using the grouper Enterospora-positive DNA sample as a template showed an amplification curve and a red band, respectively, while other pathogens and negative controls all showed negative results, indicating that the two established methods have good specificity for grouper Enterospora.

[0088] Example 7

[0089] Clinical tissue and water sample testing

[0090] RT-ERA and ERA-LFD were used to detect Enterospora in 42 tissue samples (Table 2) and 26 water samples (Table 3) collected from 2023 to 2024, and the results were compared with those of conventional PCR and basic ERA. Conventional PCR: The primers used were EEP-PAGE-F2 / EEP-PAGE-R2, and the reaction system (50 μL) contained 2 μL (10 μM) forward primer, 2 μL (10 μM) reverse primer, 2 μL template DNA, 25 μL 2× RapidTaq Master Mix, and dd water to 50 μL. DNA denaturation was performed at 95°C for 5 minutes, followed by a total of 35 PCR cycles, with the following specific conditions: DNA denaturation at 94°C for 30 seconds, primer annealing at 57°C for 30 seconds, and DNA polymerization at 72°C for 1 minute. After the last cycle, the reaction was terminated at 72°C for 10 minutes. Basic ERA: The best primers EEP-HPLC-F2 / EEP-HPLC-R2 screened out by the basic ERA in Example 1 were used; the reaction was carried out at 39° C. for 15 min.

[0091] RT-ERA: The primers used were EEP-HPLC-F2 / EEP-HPLC-R2 and the probe was EEP-probe2a; the reaction was carried out at 39°C for 16 minutes.

[0092] ERA-LFD: The primers used were EEP-HPLC-F2 / EEP-R2-biotin and the probe was EEP-Sprobe2a; the reaction was carried out at 40°C for 10 min. The positive results of the four methods of conventional PCR, basic ERA, RT-ERA and ERA-LFD are shown in Statistical Table 4. The specific gel images, fluorescence amplification curves and test strip results of each detection method are shown in Figures 6 to 9 Among them, the conventional PCR method detected 22 positive samples: 21 samples from Wenchang F1 farm (tissue samples of Wenchang emaciation), and 1 tissue sample from Danzhou F10P3 farm. The results are shown in Figure 6 A to F. ERA method detected 26 positive samples: the results are shown in Figure 7 A~F; RT-ERA method detected 29 positive samples and calculated the gene copy number of Epinephelus grouper based on Ct value: the results are shown in Figure 8 A~B; ERA-LFD method detected 26 positive samples: the results are shown in Fig. 9 A to F. 23 tissue samples from the F1 farm in Wenchang City (tissue samples of emaciated fish in Wenchang City), and 3 tissue samples and 3 water samples from the F8P1, F10P1 and F10P3 farms in Danzhou City.

[0093] The positive rate results of tissue samples and water samples were consistent with those of RT-ERA, ERA-LFD and basic ERA. The positive rate of grouper enterosporidial was 61.9% in tissue samples and 11.5% in water samples. The positive rates of tissue and water samples detected by conventional PCR were 52.4% and 0%, respectively.

[0094] Table 2. Detection results of conventional PCR, basic ERA, RT-ERA and ERA-LFD on clinical fish tissue samples

[0095]

[0096]

[0097] Note: - represents the intestinal microsporidia test result is negative; + represents the intestinal microsporidia test result is positive.

[0098] Table 3. Detection results of environmental water samples by conventional PCR, basic ERA, RT-ERA and ERA-LFD

[0099]

[0100]

[0101] Note: - represents the intestinal microsporidia test result is negative; + represents the intestinal microsporidia test result is positive.

[0102] Table 4 Statistics of RT-ERA, ERA-LFD, basic ERA and conventional PCR test results

[0103]

[0104] RT-ERA and ERA-LFD detection, the two probes are in the same position, with the same sensitivity and specificity. RT-ERA reaction is effective in the temperature range of 33-41 ° C. At 39 ° C, the Ct value is the lowest, the slope is the highest, and the fluorescence value is the largest, so 39 ° C is selected as the optimal reaction temperature. The reaction reaches a plateau within 28 to 40 cycles, so 14 to 20 min is used as the optimal reaction time range. ERA-LFD found that the optimal reaction temperature and time were 40 ° C and 10 min, respectively, based on the brightness of the red control band. The specificity of RT-ERA and ERA-LFD detection was determined by detecting common pathogens of grouper, and only the intestinal spore plasmid standard showed positive results on the amplification curve and the control band. In addition, the present invention found that the sensitivity of HPLC purification primers in the basic ERA is higher than that of PAGE purification primers.

[0105] The RT-ERA and ERA-LFD methods of the present invention have the advantages of simple operation, high efficiency and high sensitivity compared with nucleic acid amplification methods such as qPCR (Taq-Man probe method) and conventional PCR. ERA can overcome the influence of inhibitors in the sample DNA preparation process, improve the sensitivity of detection, and use plasmid standards as templates. RT-ERA combined with a portable fluorescence quantitative instrument can accurately judge the results through the quantitative curve at 39°C and a time range of about 14 to 20 minutes. The results of ERA-LFD are clearly interpreted, and the results can be obtained within 40°C and 10 minutes, which is clearer and more intuitive than the basic ERA and PCR through the band interpretation on the gel map.

[0106] The present invention detected 68 actual samples, including 28 tissue samples with severe emaciation and mild emaciation symptoms, 14 tissue samples and water samples with other clinical symptoms and health, and 12 water samples without corresponding tissue samples. The positive detection rates of RT-ERA, ERA-LFD and basic ERA showed the same results (tissue samples: 61.9%, water samples: 11.5%), which were better than the positive detection rate of conventional PCR (tissue samples: 52.4%, water samples: 0). An important reason is that ERA has a stronger antagonistic effect on inhibitors in sample DNA, so ERA is more sensitive. Compared with tissue samples with other clinical symptoms (F8 and F10), the emaciation tissue sample (F1) has a lower Ct value in RT-ERA, a higher fluorescence intensity, a brighter band in ERA-LFD, and a faster detection speed. Quantitative analysis of RT-ERA showed that in samples that have shown obvious symptoms of enterosporidial infection, the gene copy number of grouper enterosporidial was between 1.0×10 6 copies~6.0×10 6 copies, while the copy numbers of samples with other clinical symptoms and water samples were much lower than 1.0×10 6 The number of pathogen DNA copies in the sample is highly correlated with symptoms. By measuring the number of pathogen DNA copies, we can understand and analyze the disease process. This method is highly practical.

[0107] The test results of fish and water in the same farm are consistent (F8P1, F10P1 and F10P3). One possible reason is that grouper enterosporea is spread through water. Infected fish release pathogens when they come into contact with water, thereby infecting other healthy fish. On the one hand, when grouper enterosporea are detected in tissue samples, it means that the pathogen already exists in the water, but there is a lack of suitable conditions for reproduction, and the number that causes fish disease has not yet reached a pathogenic level. On the other hand, the detection of enterosporea in water samples indicates that a large number of pathogens already exist in the water, or will reach a pathogenic level, threatening healthy fish. Therefore, people need to continuously monitor the condition of water bodies and take preventive measures to reduce the losses that the aquaculture industry may face.

[0108] The present invention indirectly evaluates whether the fish carries pathogens and predicts the potential severity of the disease by detecting the concentration of pathogens in water samples, thereby providing an effective prediction and monitoring method for grouper enterosporosis infection. Through timely detection and early intervention measures, such as treating young fish and changing water bodies in a timely manner, the horizontal transmission of pathogens can be cut off and disease outbreaks can be prevented. In addition, the method can achieve the monitoring purpose through water sample detection without harming the fish, significantly reducing losses in the aquaculture industry.

Claims

1. A method for detecting grouper enterosporidia, characterized in that: The Latin name of the grouper enterospora is Enterospora epinepheli; the method comprises detecting the 18SrRNA gene sequence of the grouper enterospora by using the enzymatic constant temperature amplification technology ERA; The primer sequences of the ERA are: EEP-HPLC-F2; EEP-HPLC-R2; in the direction from the 5' end to the 3' end, the nucleotide sequence of the EEP-HPLC-F2 is sequence number (ID): 25; the nucleotide sequence of the EEP-HPLC-R2 is sequence number (ID):

26.

2. The method for detecting grouper enterosporidia according to claim 1, characterized in that: The primer is purified by high performance liquid chromatography.

3. The method for detecting grouper enterosporidia according to claim 1 or 2, characterized in that: The length of the primer is 206 bp.

4. The method for detecting grouper enterosporidia 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.

5. The method for detecting grouper enterosporidia according to claim 4, characterized in that: The real-time fluorescence quantitative binding ERA probe is EEP-probe2a; in the direction from the 5' end to the 3' end, the nucleotide sequence of the EEP-probe2a is sequence number (ID): 49; a FAM-dT fluorescent group is set at the 30th base of the 5' end of sequence number (ID): 49, and a BHQ1-dT quenching group is set at the 15th base of the 3' end, a C3-Spacer blocking group is set at the 3' end, and a tetrahydrofuran is set between the fluorescent group and the quenching group; The probe of the ERA combined lateral flow chromatography test strip is: EEP-Sprobe2a, and the nucleotide sequence of the EEP-Sprobe2a is sequence number (ID): 50; a FAM fluorescent group is set at the 5' end of sequence number (ID): 50, 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 grouper enterosporidia according to claim 5, characterized in that: The length of the probe is 46 bp.

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

8. The method for detecting grouper enterosporidia according to claim 4, characterized in that: The reaction temperature of the real-time fluorescence quantitative binding ERA is 33° C. to 41° C., preferably 39° C.; the reaction time is preferably 14 to 20 minutes.

9. The method for detecting grouper enterosporidia according to claim 4, characterized in that: The reaction temperature of the ERA combined with the lateral flow chromatography test strip is 37° C. to 40° C., preferably 40° C.; the reaction time is 5 to 35 minutes, preferably 10 minutes.

10. The method for detecting grouper enterosporidia according to claim 1 or 2, characterized in that: The detection limit of ERA is 2×10 0 copies / μL.

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