Primers and probes for detecting grouper enterospora and their applications

Abstract

The present invention belongs to the technical field of microbial detection, and specifically relates to primers and probes for detecting grouper intestinal cysts and their applications. The present invention designs primers and probes for the SSUrRNA gene of grouper intestinal cysts, establishes real-time fluorescence quantitative PCR based on TaqMan-MGB probe and a detection method based on isothermal amplification technology, and the detection sensitivity of the two methods for the target gene is 6.1 and 268 copies / reaction, respectively, and has the advantages of fast detection, specificity and good repeatability in the detection application. The isothermal amplification method can complete the detection within 20 minutes under constant temperature conditions and can visually judge the results, which is suitable for on-site diagnosis of pathogens and development of detection kits.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and particularly relates to grouper enterosporea ( Enterospora Epinepheli ) detection primers and probes and their applications. Background Art

[0002] Grouper is rich in nutrition, delicious meat and high economic value. It is an important marine aquaculture species in southern my country and Southeast Asia. In 2023, the aquaculture output of various groupers in my country reached more than 240,000 tons, with more than 50 species of 10 genera. With the increase in the degree of intensive scale, the continuous emergence of aquaculture diseases has jeopardized the healthy development of the grouper aquaculture industry.

[0003] Microsporidia are an important parasitic disease in aquaculture in recent years. Currently, four types of microsporidia have been reported in grouper, namely, Epinephelus polyphekadion ) of Arabidopsis arabicus ( Glugea arabica )、Epinephelus reticulatus( E.chlorostigma ) of Pili Pleistophora sp.), red-spotted grouper ( E.akaara ) of the grouper G.epinephelusis ) and pearl grouper ( E. fuscoguttatus ♀× E. lanceolatus ♂) of grouper enterocolitica ( Enterospora epinepheli ). Among the above four grouper microsporidians, grouper enterosporidia was recently discovered and officially named in 2018. This pathogen can cause symptoms such as anorexia, thinning of the intestinal wall, and severe weight loss in infected fish. In severe cases, it can cause a large number of deaths in infected fish. Sensitive and specific detection of pathogens is the basis of pathogen prevention and control. Currently, the molecular detection of this pathogen includes conventional PCR and fluorescent quantitative PCR based on TaqMan probes.

[0004] Taqman-MGB probe is a new type of probe in the fluorescence quantitative PCR detection method. After adding a minor groove binder (MGB) that can insert into the minor groove of DNA to its 3' end, the Tm value of the probe is effectively increased, thereby improving the specificity of sample detection. The MGB probe is labeled with a fluorescent group at the 5' end and a nonfluorescent quencher (NFQ) at the 3' end. The signal generated by the fluorescent reporter group at the 5' end is quenched by the NFQ group, which can effectively reduce the fluorescent background signal and further improve the sensitivity of the detection. Loop-mediated isothermal amplification (LAMP) is a gene isothermal amplification technology. The basic primers in this method include 4, namely F3, B3, FIP (primers F1c and F2) and BIP (primers B1c and B2). A pair of LOOP primers, namely LF (between primers F1c and F2) and LB (between primers B1c and B2), which can also initiate strand displacement DNA synthesis, can be added to the LAMP detection primers to further increase the reaction speed. However, the LOOP primers are limited by the requirements of primer design and the limited sequence interval, which makes it difficult to design. Sometimes only one of the loop primers can be designed or it cannot be designed. LAMP detection can complete the isothermal rapid amplification of genes under the conditions of a simple constant temperature device (such as a water bath or a metal bath, etc.). In the judgment of the test results, the naked eye can judge the test results by the presence or absence of white precipitate of magnesium pyrophosphate in the amplification product or the color change after adding nucleic acid dyes. Compared with conventional PCR methods, LAMP detection does not require special equipment, template denaturation, temperature cycling, electrophoresis analysis and other steps, and has the advantages of rapid detection, strong specificity and high sensitivity, which is suitable for the development of pathogen field diagnosis detection kit products. In the LAMP reaction system, by adding nucleic acid dyes such as EvaGreen® and placing it in a quantitative PCR instrument, the intensity of the fluorescent signal after the product combines with the fluorescent dye can be analyzed to achieve real-time quantitative detection of the target gene without labeled probes. At present, there is no quantitative detection and LAMP detection method based on Taqman-MGB probe for grouper enterocybe. Summary of the invention

[0005] The purpose of the present invention is to provide primers and probes for detecting grouper enterocystis and their application. The present invention extracts DNA from samples of pearl grouper infected with grouper enterocystis, and designs detection primers and probes for the small subunitribosomal RNA (SSUrRNA) gene of grouper enterocystis.

[0006] The present invention is achieved through the following technical solutions:

[0007] The detection primers and probes for grouper enterospores of the present invention include 3 sets of isothermal amplification primers and 1 set of fluorescent quantitative PCR detection primers and probes, and the nucleotide sequences thereof are as follows:

[0008] (1) The nucleotide sequences of the three sets of isothermal amplification primers are as follows:

[0009] 1) Composition and nucleotide sequence of the first set of primers:

[0010] Primer 1: E.ep_F3_1: 5′-AACACGATGATAAAAATCGTAGA-3′, as shown in SEQ ID NO: 1;

[0011] Primer 2: E.ep_B3_1: 5′-TGCTAGAATTACAGCGGTAT-3′, as shown in SEQ ID NO: 2;

[0012] Primer 3: E.ep_FIP_1:

[0013] 5′-TCCAGGTAGAGTCTAGAGATTTCATTTTTTTTGCTAAATATTGCTAGCGG-3′, as shown in SEQ ID NO: 3;

[0014] Primer 4: E.ep_BIP_1:

[0015] 5′-GAGGCGAAAGCGACGTTCTTTTTTCTAATCACTTTCAATACTCCAGC-3′, as shown in SEQ ID NO: 4;

[0016] Primer 5: E.ep_LF_1: 5′-CCTATACCACGTTCCCTATCCATT-3′, as shown in SEQ ID NO: 5;

[0017] Primer 6: E.ep_LB_1: 5′-AGACGTATCCGAGGGTCAAG-3′, as shown in SEQ ID NO:6.

[0018] 2) Composition and nucleotide sequence of the second set of primers:

[0019] Primer 1: E.ep_F3_2: 5′-GGATAGTACAGCCGCAAG-3′, as shown in SEQ ID NO: 7;

[0020] Primer 2: E.ep_B3_2: 5′-TCCAACACTTAAGTTAAAGATCA-3′, as shown in SEQ ID NO: 8;

[0021] Primer 3: E.ep_FIP_2:

[0022] 5'-CGTTGAGTTAAATTAAGCGGCATAATTTTACTGAAACTTAAACGATATTGGC-3', as shown in SEQ IDNO: 9;

[0023] Primer 4: E.ep_BIP_2:

[0024] 5'-GGAAAACTTACCAGGGTCAAGTTTTTTCATCAATTTACAACGGCCAT-3', as shown in SEQ ID NO: 10;

[0025] Primer 5: E.ep_LF_2: 5′-TCCACTACTGGTGGTAAACTTCC-3′, as shown in SEQ ID NO: 11;

[0026] Primer 6: E.ep_LB_2: 5′-CATGAGATAAACGAGAGTGGTGC-3′, as shown in SEQ ID NO:12.

[0027] 3) Composition and nucleotide sequence of the third set of primers:

[0028] Primer 1: E.ep_F3_3: 5′-ACGGTAACCTGTGGCTAA-3′, as shown in SEQ ID NO: 13;

[0029] Primer 2: E.ep_B3_3: 5′-CGTCTAATAAATGTCTCTTCTCAA-3′, as shown in SEQ ID NO: 14;

[0030] Primer 3: E.ep_FIP_3:

[0031] 5'-CGTCACCCATTGTTGCCTTGTTTTGCGCAGTCCTATTATGTTTG-3', as shown in SEQ ID NO: 15;

[0032] Primer 4: E.ep_BIP_3:

[0033] 5'-AATCAGGGTTTGATTCCGGAGAGTTTTAAGTGGACAATTTTCGCG-3', as shown in SEQ ID NO: 16;

[0034] Primer 5: E.ep_LF_3: 5′-GGAGTCCATTACACTACCAA-3′, as shown in SEQ ID NO: 17;

[0035] Primer 6: E.ep_LB_3: 5′-GGAGCCTGAGAGATGGC-3′, as shown in SEQ ID NO:18.

[0036] (2) Nucleotide sequences of primers and probes for fluorescence quantitative PCR detection:

[0037] Primer 1: E.ep_F: 5′-ACGTCCGTAGCTGTAGATGCAA-3′, as shown in SEQ ID NO: 19;

[0038] Primer 2: E.ep_R: 5′-CCTCTGTTGGTCCAGGTAGAGTC-3′, as shown in SEQ ID NO: 20;

[0039] Probe: E.ep_P: 5′-CGTGGTATAGGTAGACAAAG-3′, as shown in SEQ ID NO:21.

[0040] In the detection of grouper enterosporea by isothermal amplification technology, primers 1-4 in the first, second and third sets of primers can be used, and primers 1-6 can also be used.

[0041] The reaction procedure of isothermal amplification based on LAMP is: 60.0℃-68.4℃ constant temperature for 1min, 20-60 cycles, preferably 64.0℃-66.4℃, more preferably 65.7℃. The amount of each component in the isothermal amplification reaction system includes: the final concentration of MgSO4 (including 2.0mM Mg in 1×Isothermal amplification buffer) 2+ ) is 4.0-12.0mM, the concentration of dNTPs is 0.8-1.8mM, Bst 2.0 WarmStart ® The concentration of DNA polymerase is 0.128-0.576U / μL, the concentration of betaine is 0-1.4mM, preferably, the final concentration of MgSO4 is 6.0mM, and the concentration of dNTPs is 1.2mM. Bst 2.0 WarmStart ® The concentration of DNA polymerase is 0.512U / μL, and the concentration of betaine is 0.2mM. In the above reaction system, EvaGreen at a concentration of 0.5-2.5μM can also be added. ® Dye, preferably 0.75 μM. After the reaction is completed, the system is placed at 80°C for 5 minutes to terminate the reaction. In the amplification reaction, the DNA template of the sample to be tested, which has been denatured at 95°C, is added to the reaction system.

[0042] The isothermal amplification primers (including the first, second and third sets) can be used for qualitative and quantitative detection of pathogens, and the determination of the detection results includes the following methods:

[0043] (1) Qualitative testing:

[0044] (a) Electrophoresis: EvaGreen was not added to the reaction system ® Dye, the test can be placed in a PCR instrument, water bath or metal bath. In the case of no contamination in the negative control test, if the electrophoresis products of the positive sample and the test sample are ladder-like bands, it is considered that the pathogen test is positive, otherwise the pathogen sample test is considered negative;

[0045] (b) Visual observation: EvaGreen was not added to the reaction system ® Dye, the test can be placed in a PCR instrument, water bath or metal bath. Add 2% volume ratio of GeneFinder to the product of the amplification reaction. TM The nucleic acid dye, the amplification products of positive samples and test samples change from orange-yellow to fluorescent green, which is considered positive for pathogen detection, while the negative control and negative samples are orange-yellow; the amplification products have obvious white precipitation (magnesium pyrophosphate), which is considered positive for pathogen detection;

[0046] (c) Amplification curve analysis: EvaGreen was added to the reaction system ® Dye is placed in a quantitative PCR instrument. The test results are analyzed by amplification curves. In the case of no contamination in the negative control, the positive sample and the test sample have amplification curves, which is considered to be positive for pathogen detection.

[0047] (2) Quantitative detection: In the amplification curve analysis in the above qualitative detection, the pathogen load in the sample can be calculated based on the linear relationship between the template amount and the CT value and the CT value of the test sample. When the first set of primers for isothermal amplification is used for detection, the linear expression of the logarithmic value x of the nucleic acid copy number of the plasmid standard and the CT value y is y=-1.7195x+23.971.

[0048] The application mode of the fluorescent quantitative PCR detection primer and probe is as follows: when the probe is used, the 5' end and 3' end of the probe are modified with a fluorescent reporter group and a fluorescent quencher group, respectively. Preferably, the probe is a Taqman-MGB probe, the 5' end is modified with a fluorescent reporter group 6-carboxyfluorescein (6-FAM), and the 3' end is modified with MGB-NFQ. During the detection, the reaction temperature is 55-62°C, preferably 58°C; the final concentrations of the primer and the probe are both 0.1-0.5 μM, preferably 0.3 μM. In the judgment of the detection result of the qualitative detection, when the negative control test is free of contamination, the positive sample and the test sample have an amplification curve, which is considered to be positive for the pathogen detection. When the probe is not used, a nucleic acid fluorescent dye such as SYBRGreen Ⅰ can be added to the reaction system. If the melting curve of the positive sample and the detected amplification product is a single peak graph, the sample is considered to be positive for the pathogen detection. When the fluorescent quantitative PCR detection primers and Taqman-MGB probe are used to detect grouper enterocybe, the linear expression of the logarithmic value x of the nucleic acid copy number of the plasmid standard and the CT value y is y=-3.195x+42.413. In quantitative analysis, the pathogen load in the sample can be calculated based on the linear relationship between the template amount and the CT value and the CT value of the test sample.

[0049] The detection primers and probes for grouper enterocybe cysts of the present invention are used for the detection and quantitative analysis of grouper enterocybe cysts, and also include the development and application of detection reagents or kit products for the pathogen.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. Strong specificity

[0052] The invention determines the conservative region of the gene based on the analysis results of the SSU rRNA gene of grouper enterocystis obtained from epidemiological investigation and disclosed, and the designed primers and probes are suitable for the specific detection of different strains of grouper enterocystis.

[0053] 2. The detection is sensitive and rapid and can provide quantitative analysis results

[0054] The isothermal amplification of the present invention can complete the amplification of 2.68×10 2 -2.68×10 9 The detection of target genes in the copy range can also realize fluorescent quantitative PCR detection of pathogens without the need for labeled probes; the fluorescent quantitative PCR detection can be completed within 60 minutes, and its detection sensitivity of 6.1 copies / reaction for the target gene is better than other reported detection methods for the pathogen, and is suitable for early diagnosis and early warning of pathogens.

[0055] 3. The test operation is simple and can be used for on-site diagnosis

[0056] The primers for isothermal amplification of the present invention can complete the detection reaction under simple constant temperature conditions (such as in a water bath or a metal bath). After adding nucleic acid fluorescent dyes SYBR Green or GeneFinderTM or metal ion indicator dye calcein to the reaction product, the detection result can be judged on-site by naked eyes, which is suitable for the development of on-site diagnostic kit products. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 , comparison of the fluorescent quantitative PCR detection primers and probes of the present invention with different strains of Epinephelus grouper;

[0058] Figure 2 , standard curve of TaqMan-MGB fluorescence quantitative PCR method for Enterospora grouper;

[0059] Figure 3 , and the detection sensitivity of TaqMan-MGB fluorescence quantitative PCR for Enterospora in grouper, where 9-0 represent the concentration of 6.1×10 9 -6.1×10 0 copies / μL of pMD18-ZL plasmid, N: negative control;

[0060] Figure 4 , conventional PCR sensitivity detection diagram of grouper Enterospora, where M: DNA Marker DL 2000, lanes 7-0 represent concentrations of 6.1×10 7 -6.1×10 0 copies / μL of pMD18-ZL plasmid, N: negative control;

[0061] Figure 5 , the detection specificity of TaqMan-MGB fluorescence quantitative PCR for grouper Enterocystis, where 1: grouper Enterocystis; 2: EHP; 3: Ameson sp.; 4: Trichuris trichuris; 5: Hepatocytosis; 6: Spiroplasma; 7: White spot syndrome virus; 8: Photobacterium mermanii subspecies; 9: Edwardsiella piscicidalis; 10: Viral neuronecrosis; 11: Grouper iridovirus; N: Negative control;

[0062] Figure 6 , electrophoresis of the products of isothermal amplification of grouper Enterospora (first set of primers), where lane 1: pMD18-ZL plasmid; lanes 2-5: grouper muscle tissue genomic DNA; lane 6: negative control;

[0063] Figure 7 , the products of isothermal amplification of Epinephelus grouper (first set of primers) were added to GeneFinder TMColor changes after nucleic acid dyeing, where 1: pMD18-ZL plasmid; 2-7: grouper muscle tissue genomic DNA; 8: negative control;

[0064] Figure 8 , isothermal amplification curve of grouper enterocolitica based on LAMP method, among which 1-8: 2.68×10 9 , 2.68×10 8 , 2.68×10 7 , 2.68×10 6 , 2.68×10 5 , 2.68×10 4 , 2.68×10 3 and 2.68×10 2 copies of pMD18-ZL plasmid standard; 9: 2.68×10 1 copy; 10: negative control;

[0065] Fig. 9 , the standard curve of isothermal amplification of Epinephelus grouper using the pMD18-ZL plasmid standard as the template;

[0066] Fig.10 , specific detection analysis of isothermal amplification of grouper enterococci, wherein, 1-15: pathogen-positive nucleic acids 1-15 in Table 3; 16: negative control. DETAILED DESCRIPTION

[0067] To further illustrate the method and effect of the present invention, the present invention is further described below in conjunction with examples. Unless otherwise specified, the drugs and materials used in the following examples are all commercially available.

[0068] Example 1: Sample collection and analysis of grouper enterosporea

[0069] In 2021 and 2023, multiple batches of diseased pearl grouper samples were collected from Guangdong Province. EnterF125 / EnterR1153 primers (Xu LW, et al. Outbreak of enteric microsporidiosis of hatchery-bred juvenile groupers, Epinephelus spp., associated with a new intranuclear microsporidian in China [J]. Journal of Fish Diseases, 2016.40 (2): 183-189) were used to perform PCR detection and sequencing analysis of the SSUrRNA gene of Epinephelus spp., and it was determined that the diseased fish tissues were positive for Epinephelus spp. Based on the sequencing results of Epinephelus spp. obtained and the SSUrRNA gene sequence analysis of other published strains of the pathogen, the conserved nucleotide sequence of the gene was determined, and the subsequent detection primers and probes were designed based on this.

[0070] Example 2: Primers and probes for the Taqman-MGB fluorescence quantitative PCR detection method for grouper enterococci

[0071] According to the conservative nucleic acid sequence of SSUrRNA gene, the nucleotide sequences of the designed fluorescent quantitative PCR detection primers and probes are as follows:

[0072] Primer 1: E.ep_F: 5'-ACGTCCGTAGCTGTAGATGCAA-3',

[0073] Primer 2: E.ep_R: 5'-CCTCTGTTGGTCCAGGTAGAGTC-3',

[0074] Probe: E.ep_P: 5'-CGTGGTATAGGTAGACAAAG-3'.

[0075] The nucleotide sequences of the primers and probes designed in the present invention were compared and analyzed with different strains of Epinephelus ocellatus (including different strains collected by our epidemiology and published Epinephelus ocellatus) using MEGA software. Figure 1 The results show that the primers and probes of the present invention are suitable for the detection of different strains of grouper Enterospora, which is different from the situation that the probes and primers in the quantitative detection of the pathogen have not matched the corresponding nucleotide sequences of the target gene.

[0076] The probe 5' in this embodiment is modified with the fluorescent reporter group 6-FAM, and the probe 3' is modified with MGB-NFQ. In addition, the probe 5' can also be modified with other fluorescent reporter groups, such as TET, JOE, VIC, NED, CY3, HEX, etc., and the probe 3' can also be modified with other fluorescent quenching groups, such as TAMRA, BHQ, etc. In the test results, if there is no contamination in the negative control test, and there is an amplification curve in the positive control and the test sample, the sample is considered to be positive for pathogen detection. The primers E.ep_F and E.ep_R can also be used for dye-based fluorescent quantitative PCR. No probe is added to the reaction system, but nucleic acid fluorescent dyes such as SYBRGreen Ⅰ are added. There is no contamination in the negative test, and the melting curve in the test results of the positive control and the test sample is a single peak graph, which is considered to be positive for the sample pathogen detection. In the above two detection methods, the equation can be determined based on the linear relationship between the template amount and the CT value, and the pathogen load in the sample can be calculated by detecting the CT value of the sample.

[0077] Example 3: Construction of recombinant plasmid standards for detection of grouper enterospora

[0078] The nucleic acid of the pathogen tissue was amplified using primers EnterF125 / EnterR1153 of Xu et al. (2016). The PCR product was purified by gel recovery using a gel recovery kit (Tiangen, China) and then ligated with pMD18-T vector (TaKaRa, Dalian, China) to construct the recombinant plasmid pMD18-ZL and transformed into Escherichia coli DH5α strain was plated on LB solid medium containing Amp and cultured overnight at 37°C. The positive colonies were screened with M13 primers and sequenced. The bacterial solution containing the positive recombinant plasmid pMD18-ZL was cultured overnight at 37°C. The recombinant plasmid pMD18-ZL was extracted using a plasmid miniprep kit (Tiangen, China). The plasmid concentration was determined using Nanodrop 2000 c (Thermo) and gradient diluted to 6.1×10 9 -6.1×10 0 copies / μL, stored at -20℃ for future use.

[0079] Example 4: Optimization of primer and probe concentrations for Taqman-MGB fluorescent quantitative PCR detection of grouper enterococci

[0080] In quantitative PCR detection, the reaction system is Premix Ex Taq™ (Probe qPCR) kit (TaKaRa) instructions. During the optimization, the primers and probes of the present invention were evaluated at final concentrations of 0.1μM, 0.2μM, 0.3μM, 0.4μM, and 0.5μM, respectively, and the detection effect was evaluated by detecting the CT value and the fluorescence intensity increase value (ΔRn). The 20μL reaction system contained 10μL of Premix Ex Taq (Probe qPCR), primers E.ep_F, E.ep_R and probe E.ep_P with different final concentrations (0.1-0.5μM), 0.2μL of ROX Reference Dye (50×), and 1μL of plasmid standard as template, and the amplification reaction was carried out in the AppliedBiosystems QuantStudio 3 (Thermo Fisher Scientific, USA) instrument. The reaction program was 95℃ 30s, 95℃ 5s and X℃ 30s (X is the annealing temperature gradient of 55-62℃), and 40 cycles. The final concentrations of the optimized primers E.ep_F, E.ep_R and probe E.ep_P were all 0.3 μM, and the annealing temperature was 58°C. This reaction concentration and temperature were used in subsequent experiments.

[0081] Example 4: Standard curve, detection sensitivity and repeatability evaluation of Taqman-MGB fluorescence quantitative PCR detection method for grouper enterocystis

[0082] (1) Standard curve

[0083] The logarithm and CT value of the plasmid standard copy number pMD18-ZL, when the template concentration is 6.1×10 9 -6.1×10 0 There is a good linear relationship in the copies / μL range, and the linear equation is y=-3.195x+42.413, R 2 =0.999, the amplification efficiency is 105.6%, and the amplification efficiency is good. Figure 2 .

[0084] (2) Sensitivity

[0085] The pMD18-ZL plasmid standard (6.1×10 9 -6.1×10 0 The results showed that the detection sensitivity of the Taqman-MGB fluorescent quantitative PCR for grouper enterocybe was 100 times that of the conventional PCR of Xu et al. (2016, see Example 1). The detection sensitivity of the two methods was improved by 6.1 copies / reaction ( Figure 3 ) and 6.1×10 2 copies / reaction ( Figure 4 ). The detection sensitivity of the fluorescent quantitative PCR method of the present invention is also better than the reported quantitative detection method of the pathogen of 10 copies / reaction (Quan-lin Liu, et al. Establishment of a TaqMan probe-based qPCR assay for detecting microsporidia Enterosporaepinepheli in grouper[J]. Journal of Fish Diseases, 2024, 7 (3)), and is suitable for early monitoring, early warning and prevention and control of the pathogen.

[0086] (3) Repeatability

[0087] In the repeatability experiment of the detection method, 6.1×10 9 -6.1×10 0 The pMD18-ZL plasmid with 10 copies / μL was used as a template for intra-batch and inter-batch detection. Three replicates were set for each concentration. The coefficients of variation within and between batches were in the range of 0.22%-1.80% and 0.21%-1.95%, respectively (Table 1). The results showed that the detection method had good repeatability and could meet the requirements of clinical detection.

[0088] Table 1 Intra-assay and inter-assay reproducibility of TaqMan-MGB fluorescence quantitative PCR method for Enterospora spp. in grouper

[0089]

[0090] Example 5: Specificity evaluation of the Taqman-MGB fluorescence quantitative PCR detection method for grouper enterocystis

[0091] In the specific detection, shrimp hepatoenterostomyces ( Enterocytozoon Hepatopenaei , EHP), marine crab microsporidia Ameson sp., blood egg whipworm ( Hematodinium perezi )、Cnidaria hepatica( Hepatosporaeriocheir ), Spiroplasma ( Spiroplasma eriocheiris ), White Spot Syndrome Virus (WSSV), Photorhabdus mermanii subspecies Photobacterium damselae subsp. damselae ), Edwardsiella piscicida ( Edwardsiella piscicida), viral nervous necrosis (VNN), and Singapore grouper iridovirus (SGIV) DNA were used as qPCR templates, and only the pMD18-ZL plasmid of Echinocystis grouper was positive ( Figure 5 ).

[0092] Example 7: Design of primers for isothermal amplification of Epinephelus spp.

[0093] Based on the conserved region of the SSUrRNA gene of Epinephelus grouper and the sequence analysis results of the gene of the pathogen that we collected and sequenced, we designed three sets of detection primers for isothermal amplification, each set of 6 primers as follows:

[0094] (1) Composition and nucleotide sequence of the first set of primers:

[0095] Primer 1: E.ep_F3_1: 5'-AACACGATGATAAAAATCGTAGA-3',

[0096] Primer 2: E.ep_B3_1: 5'-TGCTAGAATTACAGCGGTAT-3',

[0097] Primer 3: E.ep_FIP_1:

[0098] 5'-TCCAGGTAGAGTCTAGAGATTTCATTTTTTTTGCTAAATATTGCTAGCGG-3',

[0099] Primer 4: E.ep_BIP_1:

[0100] 5'-GAGGCGAAAGCGACGTTCTTTTTTCTAATCACTTTCAATACTCCAGC-3',

[0101] Primer 5: E.ep_LF_1: 5'-CCTATACCACGTTCCCTATCCATT-3',

[0102] Primer 6: E.ep_LB_1: 5′-AGACGTATCCGAGGGTCAAG-3′.

[0103] (2) Composition and nucleotide sequence of the second set of primers:

[0104] Primer 1: E.ep_F3_2: 5'-GGATAGTACAGCCGCAAG-3',

[0105] Primer 2: E.ep_B3_2: 5'-TCCAACACTTAAGTTAAAGATCA-3',

[0106] Primer 3: E.ep_FIP_2:

[0107] 5'-CGTTGAGTTAAATTAAGCGGCATAATTTTACTGAAACTTAAACGATATTGGC-3',

[0108] Primer 4: E.ep_BIP_2:

[0109] 5'-GGAAAACTTACCAGGGGTCAAGTTTTTTCATCAATTTACAACGGCCAT-3',

[0110] Primer 5: E.ep_LF_2: 5'-TCCACTACTGGTGGTAAACTTCC-3',

[0111] Primer 6: E.ep_LB_2: 5′-CATGAGATAAACGAGAGTGGTGC-3′.

[0112] (3) Composition and nucleotide sequence of the third set of primers:

[0113] Primer 1: E.ep_F3_3: 5'-ACGGTAACCTGTGGCTAA-3',

[0114] Primer 2: E.ep_B3_3: 5'-CGTCTAATAAATGTCTCTTCTCAA-3',

[0115] Primer 3: E.ep_FIP_3:

[0116] 5'-CGTCACCCATTGTTGCCTTGTTTTGCGCAGTCCTATTATGTTTG-3',

[0117] Primer 4: E.ep_BIP_3:

[0118] 5'-AATCAGGGTTTGATTCCGGAGAGTTTTAAGTGGACAATTTTCGCG-3',

[0119] Primer 5: E.ep_LF_3: 5′-GGAGTCCATTACACTACCAA-3′,

[0120] Primer 6: E.ep_LB_3: 5′-GGAGCCTGAGAGATGGC-3′.

[0121] Example 8: Optimization and application of isothermal amplification reaction of grouper enterocolitica

[0122] Taking the pearl grouper enterospore-positive tissue DNA as the template, the reaction system and reaction temperature of the first set of isothermal amplification primers were optimized, and a temperature gradient of 60°C, 60.7°C, 62°C, 64°C, 66.4°C, and 68.4°C was set in a real-time fluorescence quantitative PCR instrument (Bio-Rad, California, the United States). The reaction was carried out for 60 min (60 cycles, 1 min per cycle). The more suitable amplification reaction temperature range in this temperature range was 64.0°C-66.4°C, and a temperature gradient of 64.0°C, 64.1°C, 64.4°C, 64.8°C, 65.3°C, 65.7°C, 65.9°C, and 66.0°C was further set. Preferably, the reaction temperature was 65.7°C.

[0123] The 25 μL LAMP reaction system contained 2.5 μL 10×Isothermal amplification buffer (containing 2 mM Mg 2+ )、20μM E.ep_FIP / E.ep_BIP 2μL、10μM E.ep_F3 / E.ep_B3 0.5μL、20μM E.ep_LF / E.ep_LB 1μL、final concentration of MgSO4 (including 2.0mM Mg in 1×Isothermal amplification buffer 2 + ) is 4.0-12.0mM, the concentration of dNTPs is 0.8-1.8mM, Bst 2.0 WarmStart ® The concentration of DNA polymerase is 0.128-0.576U / μL, the concentration of betaine is 0-1.4mM, preferably, the final concentration of MgSO4 is 6.0mM, and the concentration of dNTPs is 1.2mM. Bst 2.0 WarmStart ® The concentration of DNA polymerase is 0.512U / μL, and the concentration of betaine is 0.2mM. EvaGreen can also be added to the above reaction system. ® The dye concentration is 0.5-2.5 μM, preferably 0.75 μM. After the reaction is completed, the system is placed at 80°C for 5 minutes to terminate the reaction. In the amplification reaction, the DNA template of the sample to be tested, which has been denatured at 95°C, is added to the reaction system. The primers used in the above isothermal amplification reaction can also be primers 1-4.

[0124] Isothermal amplification primer application:

[0125] (1) Qualitative testing:

[0126] (a) Electrophoresis: EvaGreen is not added to the reaction system ® Dye, the test can be placed in a PCR instrument, water bath or metal bath. In the case of no contamination in the negative control test, if the electrophoresis products of the positive sample and the test sample are ladder-like bands, it is considered that the pathogen test is positive, otherwise the pathogen sample test is considered negative;

[0127] (b) Visual observation: EvaGreen was not added to the reaction system ® Dye, the test can be placed in a PCR instrument, water bath or metal bath. Add 2% volume of amplification reaction product such as GeneFinder TM The nucleic acid dye of the positive sample and the test sample changes from orange to fluorescent green, which is considered a positive pathogen detection, while the negative control and negative sample are orange ( Figure 6 ); If there is obvious white precipitation (magnesium pyrophosphate) in the amplification product, it is considered as positive for pathogen detection;

[0128] (c) Amplification curve analysis: EvaGreen was added to the reaction system ® Dye is placed in a quantitative PCR instrument. The test results are analyzed by amplification curves. In the case of no contamination in the negative control, the positive sample and the test sample have amplification curves, which is considered to be positive for pathogen detection ( Figure 7 ).

[0129] (2) Quantitative detection: In the amplification curve analysis of the above-mentioned qualitative detection, the pathogen load in the sample can be calculated based on the linear relationship between the template amount and the CT value and the CT value of the test sample.

[0130] Example 9: Detection sensitivity of isothermal amplification of Epinephelus spp.

[0131] Using the optimized reaction temperature and system (Example 8), 2.68×10 0 -2.68×10 9 The recombinant plasmid pMD18-ZL was used as the template (3 copies / reaction). The detection sensitivity of the first set of primers for isothermal amplification was 2.68×10 2 Copy / React ( Figure 8 ), according to the correlation between the logarithmic value (x) of the plasmid nucleic acid copy number and its corresponding CT value (y), the standard curve of the fluorescence quantitative PCR detection method for the isothermal amplification of the SSU rRNA gene of grouper Enterospora can be obtained: y=-1.7195x+23.971, R 2 =0.9962( Fig. 9 ).

[0132] Example 10: Repeatability evaluation of isothermal amplification detection results of grouper enterosporea

[0133] 2.68×10 2 -2.68×10 9 The reproducibility of the detection method was evaluated using the recombinant plasmid standard pMD18-ZL with the highest number of copies / reactions as a template. The coefficient of variation (CV) of the isothermal amplification (first set of primers) detection within and between batches was 0.22%-3.52% and 0.24%-1.70%, respectively (Table 2), which can provide good and stable detection results in the detection.

[0134] Table 2 Evaluation of repeatability of LAMP detection of grouper enterosporea

[0135]

[0136] Example 11: Specific detection of microsporidia in pearl grouper by isothermal amplification

[0137] The specificity test results of isothermal amplification (first set of primers) showed that this set of primers had no cross-reaction with other pathogens except microsporidia of pearl grouper (Table 3) in isothermal amplification detection ( Fig.10 ), which can provide specific detection results in detection applications.

[0138] Table 3 Pathogens used in isothermal amplification specificity detection of grouper Enterospora

[0139]

[0140] Example 12: Quantitative Detection and Analysis of Clinical Samples Using Isothermal Amplification Primers for Enterococcus grouper

[0141] The isothermal amplification primers (first set of primers) based on the LAMP method of the present invention were used to detect Enterococcus epinephrine in clinical samples, and the results of quantitative analysis are shown in Table 4. The results show that it can be used for the detection and quantitative analysis of clinical samples.

[0142] Table 4 Quantitative analysis of pathogens in samples by isothermal amplification of grouper enterosporea

[0143]

Claims

1. A primer and probe for detecting grouper enterosporea, characterized in that: The nucleotide sequences of the primers and probes are as follows: (1) Nucleotide sequence of isothermal amplification primers: Primer 1: E.ep_F3_1: 5'-AACACGATGATAAAAATCGTAGA-3', Primer 2: E.ep_B3_1: 5'-TGCTAGAATTACAGCGGTAT-3', Primer 3: E.ep_FIP_1: 5'-TCCAGGTAGAGTCTAGAGATTTCATTTTTTTTGCTAAATATTGCTAGCGG-3', Primer 4: E.ep_BIP_1: 5'-GAGGCGAAAGCGACGTTCTTTTTTCTAATCACTTTCAATACTCCAGC-3', Primer 5: E.ep_LF_1: 5'-CCTATACCACGTTCCCTATCCATT-3', Primer 6: E.ep_LB_1: 5′-AGACGTATCCGAGGGTCAAG-3′; (2) Fluorescence quantitative PCR detection primer and probe nucleotide sequence: Primer 1: E.ep_F: 5'-ACGTCCGTAGCTGTAGATGCAA-3', Primer 2: E.ep_R: 5'-CCTCTGTTGGTCCAGGTAGAGTC-3', Probe: E.ep_P: 5'-CGTGGTATAGGTAGACAAAG-3'.

2. An application of the primers and probes for detecting grouper enterospores according to claim 1, characterized in that: Detection of Enterospora in grouper for non-disease diagnostic and therapeutic purposes.

3. The use of primers and probes for detecting grouper enterospores as claimed in claim 2, characterized in that: When the isothermal amplification primers are used to detect grouper enterospora, the primers used are primers 1-4 or primers 1-6.

4. The use of primers and probes for detecting Enterospora grouper as claimed in claim 3, characterized in that: The reaction temperature is 60.0°C-68.0°C, the concentration of MgSO4 in the reaction system is 4.0-12.0 mM, the concentration of dNTPs is 0.8-1.8 mM, the concentration of DNA polymerase is 0.128-0.576 U / μL, and the concentration of betaine is 0.0-1.4 mM.

5. The use of primers and probes for detecting grouper enterospores as claimed in claim 4, characterized in that: The reaction system also includes EvaGreen at a final concentration of 0.5-2.5 μM ® Dye.

6. The use of primers and probes for detecting grouper enterospores as claimed in claim 3, characterized in that: When the isothermal amplification primers are used for detection, the linear expression of the logarithmic value x of the nucleic acid copy number of the plasmid standard and the CT value y is y=-1.7195x+23.

971.

7. The use of primers and probes for detecting grouper enterospores as claimed in claim 2, characterized in that: When fluorescent quantitative PCR detection primers and probes are used to detect grouper enterospores, the 5' end and 3' end of the probe are modified with a fluorescent reporter group and a fluorescent quencher group respectively.

8. The use of primers and probes for detecting grouper enterospores as claimed in claim 7, characterized in that: The final concentrations of primers and probes were 0.1-0.5 μM.

9. The use of primers and probes for detecting grouper enterospores as claimed in claim 7, characterized in that: When the fluorescent quantitative PCR detection primers and probes are used to detect grouper enterosporea, the linear expression of the logarithmic value x of the nucleic acid copy number of the plasmid standard and the CT value y is y=-3.195x+42.

413.

10. The use of primers and probes for detecting Enterospora grouper according to claim 2, characterized in that: Used for preparing detection reagent or kit product for grouper enterococcus.

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