Primers and probes for detecting microsporidia in crustaceans Ameson spp. and their applications

Abstract

The present invention belongs to the technical field of microbial detection, and particularly relates to crustaceans Amazon spp. Microsporidia detection primers and probes and their applications. Amazon spp., provides a conserved nucleic acid region for the SSU rRNA gene Amazon spp. detection. The detection sensitivity of the fluorescent quantitative PCR using the primers and probes of the present invention is 4.58 copies / reaction, which has high sensitivity and strong specificity. The detection primers and detection method of isothermal amplification provided by the present invention can complete the on-site rapid diagnosis of samples under simple conditions, and has Amazon spp. Development and application prospects of detection kits.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and particularly relates to crustaceans Ameson Primers and probes for detecting microsporidia spp. and their applications. Background Art

[0002] Microsporidia are intracellular, obligate fungal parasites that can infect almost all vertebrates and invertebrates. More than 180 genera and about 1,500 species of microsporidia have been discovered, of which more than 80 genera can infect aquatic organisms, more than 40 genera can infect crustaceans, and about 8 genera can infect crabs, namely Abelspora , Ameson ( Nadelspora )、 Areospora , Enterospora , Hepatospora , Nosema , Ormieresia and Thelohania .

[0003] Sarcospora Ameson portunus ) is classified as Fungi, Microsporidia, Microsporea, Microsporida, Pereziidae, Ameson Belong. Ameson Microsporidia have been reported to A. michaelis , A. metacarcini , Sarcospora A. portunus )、 A. pulvis , A. iseebi and A. herrnkindi , and species that have not yet been identified Ameson sp.. Myosporidae has now become a common nematode species in the swimming crab ( Portunus trituberculatus ) and Scylla pseudoburrowing Scylla paramamosain). When the swimming crab is seriously infected with myocytosis, it can cause typical symptoms of muscle whitening. Crabs with this symptom are also called "toothpaste crabs". Among the myocytosis detection methods, Chen Tiantian et al. (2017) established a quantitative PCR detection method based on SYBR Green I dye, with a detection sensitivity of 20 copies / reaction (Chen Tiantian, Fang Wenhong, Wang Yuan, et al. Establishment and application of SYBR Green I real-time quantitative PCR detection method for myocytosis in swimming crabs [J]. Southern Fisheries Science, 2017, 13 (6): 22-29.); The diagnostic procedures for myocytosis in swimming crabs (aquatic industry standard, SC / T 7239-2020) published the nested PCR method for myocytosis detection. Both methods are based on primers designed based on the SSU rRNA gene of the pathogen.

[0004] From 2020 to 2023, we conducted an epidemiological survey of marine crabs in the coastal areas of Shandong Province (mainly in Rizhao and Weifang areas) and discovered a variety of new Ameson sp., whose SSU rRNA gene is similar to that of the published Ameson sp. have a high degree of homology, and the above-mentioned two reported detection methods for myocytosis are not able to specifically detect the pathogen. There is no specific drug for microsporidial fungi, and such non-specific detection results are not conducive to the development of disease prevention. Ameson sp. has a large number of species and several new species have been discovered. In addition, the pathogens of this genus (such as Sarcospora) cause serious parasitic damage to crustaceans. Ameson spp. is necessary for the healthy aquaculture of crustaceans.

[0005] Among the quantitative detection methods of pathogens, the DNA-binding dye method (such as SYBR Green I, etc.) has the disadvantage of low specificity compared to the chemical method based on probes (such as TaqMan probes, etc.). In recent years, Taqman-MGB probe, as a new type of probe, can further effectively improve the specificity and sensitivity of detection in quantitative detection compared to TaqMan probe. Compared with the detection methods that require expensive professional equipment, loop-mediated isothermal amplification technology (LAMP) is a detection method that can quickly complete the detection reaction under simple constant temperature conditions (such as water bath or metal bath, etc.). This method can also add nucleic acid dyes to the reaction product to judge the test results by naked eye through color changes, which is suitable for on-site diagnosis of clinical samples. Summary of the invention

[0006] The object of the present invention is to provide a crustacean Ameson spp. Microsporidia detection primers and probes and their applications, the present invention is aimed at AmesonThe nucleotide sequence of the SSU rRNA gene of spp. is designed as follows: (1) 4 sets of primers and probes for fluorescence quantitative PCR detection (each set includes 2 primers and 1 probe); (2) 4 sets of primers for isothermal amplification (each set includes 4-6 primers). The primers and probes can be used Ameson spp., can also be used for Ameson The discovery of new species of sp.

[0007] The present invention is achieved through the following technical solutions:

[0008] Crustaceans according to the present invention Ameson The nucleotide sequences of the primers and probes for detecting Microsporidia spp. are as follows:

[0009] (1) The nucleotide sequences of the four sets of fluorescence quantitative PCR detection primers and probes are as follows:

[0010] Nucleotide sequences of the first set of primers and probes:

[0011] 1) Primer 1 Ameson- F: 5′-ATTTGGTGTCAGCTAATGGTATGG-3′, as shown in SEQ ID NO: 1;

[0012] 2) Primer 2 Ameson- R: 5'-TTGTCAGTGCTTTCGCCTTTG-3', as shown in SEQ ID NO: 2;

[0013] 3) Probe Ameson -Probe: 5'--TGAAGTCTGAGGATCCA-3', as shown in SEQ ID NO: 3.

[0014] Nucleotide sequences of the second set of primers and probes:

[0015] 1) Primer 1 Ameson- F: 5′-ATTTGACTCAACGCGGGAAA-3′, as shown in SEQ ID NO: 4;

[0016] 2) Primer 2 Ameson- R: 5'-GCCATGCACCACCATTTTAC-3', as shown in SEQ ID NO: 5;

[0017] 3) Probe Ameson -Probe: 5'-ACCAAGCTTATTTATTCAACG-3', as shown in SEQ ID NO:6.

[0018] The nucleotide sequences of the third set of primers and probes are:

[0019] 1) Primer 1 Ameson- F: 5'-GTTGCGGTTAAAACGTCCGT-3', as shown in SEQ ID NO: 7;

[0020] 2) Primer 2 Ameson- R: 5'-TTTCCAGCTTGCTACTGCCC-3', as shown in SEQ ID NO: 8;

[0021] 3) Probe Ameson -Probe: 5'-CTGACCTGTAATCTATTTGG-3', as shown in SEQ ID NO:9.

[0022] The nucleotide sequences of the fourth set of primers and probes are:

[0023] 1) Primer 1 Ameson- F: 5′-ACTGGAGGGAAAGTCTGGTG-3′, as shown in SEQ ID NO: 10;

[0024] 2) Primer 2 Ameson- R: 5'-CGCGACTACGGACGTTTTAA-3', as shown in SEQ ID NO: 11;

[0025] 3) Probe Ameson -Probe: 5'-CAGCAGCCGCGGTA-3', as shown in SEQ ID NO:12.

[0026] Preferably, in the fluorescent quantitative PCR detection method, primers and probes can be used simultaneously or only primers can be used. When using probes, Ameson- The 5' and 3' ends of the probe are labeled with a fluorescent reporter group and a fluorescent quencher group, respectively; more preferably, the probe can be a Taqman-MGB probe, with the 5' end modified with a fluorescent reporter group 6-carboxyfluorescein (6-FAM) and the 3' end modified with MGB (minor groove binder)-NFQ (nonfluorescent quencher). When the probe is not used, except for the primer Ameson- F / Ameson- In addition to R, fluorescent nucleic acid dyes can be added.

[0027] The final concentration of primers and probes in the detection system is 0.1-0.5 μM. Ameson -F / Ameson- R and Ameson The final concentrations of -Probe were 0.2 μM and 0.3 μM, respectively.

[0028] The reaction procedure of fluorescent quantitative PCR is: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, reaction temperature at 58-63°C for 30s, for a total of 40 cycles, preferably, the reaction temperature is 60°C.

[0029] Test result judgment: In the fluorescent quantitative PCR test using the probe, when the negative control is not contaminated and the sample has an amplification curve, it is considered as the sample Ameson spp. tested positive; in the fluorescent quantitative PCR test without using a probe, the melting curve of the sample was a single peak, which was considered as the sample Ameson spp. The test was positive. In quantitative analysis, the pathogen load in the sample can be calculated based on the linear relationship between the template and the CT value and the CT value of the test sample.

[0030] The first set of fluorescent quantitative PCR primers based on Taqman-MGB probe Ameson- F. Ameson- R and probe Ameson -Probe detection in samples, using plasmid standards as templates, the linear expression of the logarithmic value (x) of the nucleic acid copy number and the CT value (y) is y=-3.119x+38.978.

[0031] (2) The nucleotide sequences of the four sets of isothermal amplification detection primers are as follows:

[0032] Nucleotide sequences of the first set of primers:

[0033] 1) Primer 1 Ameson -F3: 5'-CGGTAATACCAGCTCCAGGA-3', as shown in SEQ ID NO: 13;

[0034] 2) Primer 2 Ameson -B3: 5'-GCCTCTGTCCTTGATCAACA-3', as shown in SEQ ID NO: 14;

[0035] 3) Primer 3 Ameson -FIP:

[0036] 5′-TCCAGCTTGCTACTGCCCCTATTTTTTTAAAACGTCCGTAGTCGCG-3′, as shown in SEQ ID NO: 15;

[0037] 4) Primer 4 Ameson -BIP:

[0038] 5'-AGCTAATGGTATGGGGAGGGGTTTTTGCTTTCGCCTTTGCTCCTC-3', as shown in SEQ ID NO: 16;

[0039] 5) Primer 5 Ameson -LF: 5′-AAATAGATTACAGGTCAGTCCCAAG-3′, as shown in SEQ ID NO: 17.

[0040] Nucleotide sequences of the second set of primers:

[0041] 1) Primer 1 Ameson -F3: 5'-GCTTGGGACTGACCTGTA-3', as shown in SEQ ID NO: 18;

[0042] 2) Primer 2 Ameson -B3: 5'-CGTATTCGATCCTCTAGCCT-3', as shown in SEQ ID NO: 19;

[0043] 3) Primer 3 Ameson -FIP:

[0044] 5′-CATACCATTAGCTGACACCAAATTGTTTTGTCAACAGATAGATAGGGGC-3′, as shown in SEQ ID NO: 20;

[0045] 4) Primer 4 Ameson -BIP:

[0046] 5′-GAGGGGTGAAGTCTGAGGATTTTTTTGATCAACAGAATCAATCTTTGTC-3′, as shown in SEQ ID NO: 21;

[0047] 5) Primer 5 Ameson -LF: 5'-TCTTTTCCAGCTTGCTACT-3', as shown in SEQ ID NO: 22;

[0048] 6) Primer 6 Ameson -LB: 5'-CCATGCAGGAGGAGCA-3', as shown in SEQ ID NO:23.

[0049] The nucleotide sequences of the third set of primers:

[0050] 1) Primer 1 Ameson -F3: 5'-GATCGAATACGATTAGATACCG-3', as shown in SEQ ID NO: 24;

[0051] 2) Primer 2 Ameson -B3: 5'-TTGGTAAGTTTTCCCGCG-3', as shown in SEQ ID NO: 25;

[0052] 3) Primer 3 Ameson -FIP:

[0053] 5′-TCCCCCCGGAGATCTATTTTGATTTTTTAGTAGTTCTAGCAGTGACCG-3′, as shown in SEQ ID NO: 26;

[0054] 4) Primer 4 Ameson -BIP:

[0055] 5′-GTACATGCGCAAGCAAGAAACTTTTTTGAGTCAAATTAAGCCGAAC-3′, as shown in SEQ ID NO: 27;

[0056] 5) Primer 5 Ameson -LB: 5'-AAAGAAATTGACGGAAGACTACCAC-3', as shown in SEQ ID NO:28.

[0057] The nucleotide sequences of the fourth set of primers are:

[0058] 1) Primer 1 Ameson -F3: 5'-GTAAAATGGTGGTGCATGG-3', as shown in SEQ ID NO: 29;

[0059] 2) Primer 2 Ameson -B3: 5'-TGTTCATGAGTGCTTTTCAG-3', as shown in SEQ ID NO: 30;

[0060] 3) Primer 3 Ameson -FIP:

[0061] 5′-TCTTTCGAACAAGCTATTCTGAGATTTTTCAGATGGAGTGATTTTGTGAT-3′, as shown in SEQ ID NO: 31;

[0062] 4) Primer 4 Ameson -BIP:

[0063] 5′-TGCACGCGCAATACAATGTTTTTTCCCTCAATCCCTCATGTT-3′, as shown in SEQ ID NO:32.

[0064] Preferably, the reaction system of isothermal amplification detection includes the following components in 25 μL: 10×Isothermal amplification buffer 2.5 μL, MgSO4 with a final concentration of 4.0-12.0 mM, betaine with a final concentration of 0.1-1.4 mM, dNTPs with a final concentration of 0.8-1.8 mM, 20 μM Ameson -FIP / Ameson -BIP 2 μL, 10 μM Ameson -F3 / Ameson -B3 0.5 μL, 20 μM Ameson -LF / Ameson -LB 1 μL, final concentration 0.128-0.576U / uL Bst 2.0 WarmStart ® DNA polymerase (8000U / mL, New England BioLabs), Ameson spp. DNA template 1μL, and add water to 25μL, in addition, 0.5-2.5μM 20×EvaGreen can be added to the system ® Dye (Biotium) 1 μL. The reaction temperature is 60.0℃-68.0℃, placed in a real-time fluorescence quantitative PCR instrument, and reacted for 60 minutes (60 cycles, 1 minute each cycle).

[0065] When the isothermal amplification detection primers are used for detection, the detection method is isothermal amplification or fluorescent quantitative PCR, and the following primers in each set of isothermal amplification primers can be used:

[0066] Ameson -F3, Ameson -B3, Ameson -FIP and Ameson -BIP;

[0067] You can also use Ameson -F3, Ameson -B3, Ameson -FIP, Ameson -BIP and Ameson -LF;

[0068] You can also use Ameson -F3, Ameson -B3, Ameson -FIP, Ameson -BIP and Ameson -LB;

[0069] You can also use Ameson -F3, Ameson -B3, Ameson -FIP, Ameson -BIP, Ameson -LF and Ameson -LB.

[0070] In the above LAMP assay, EvaGreen was added ® The dye system is placed in a quantitative PCR instrument. The detection reaction system and temperature can be optimized based on the low CT value and reagent cost in the amplification. In this test, the linear relationship between the template amount and the CT value can be used to optimize the detection sample. Ameson sp. loading for quantitative analysis; no EvaGreen added ® When dyeing, the reaction conditions can be optimized based on the bright bands of the product ladder electrophoresis bands and the reagent cost. In this test, 2% volume of nucleic acid dye (such as GeneFinder TM ), on-site diagnosis of samples under simple isothermal conditions is performed through color changes in the product tube.

[0071] The primers and probes of the present invention can be used for non-disease diagnosis and treatment purposes. Ameson spp., and can also be used for the development of detection reagents or kit products, and for new Ameson The discovery of sp.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] 1. The present invention has the following new findings based on a large number of epidemiological studies:

[0074] 1) Discover multiple Ameson sp., discovered and determined Ameson spp., designed and provided a conserved region of the SSU rRNA gene that can effectively carry out Ameson spp. Detection primers and probes. 2) The present invention can be applied to Ameson The discovery of new species of sp.

[0075] 2. High sensitivity and strong specificity:

[0076] The detection sensitivity of 4.58 copies / reaction of the fluorescent quantitative PCR of the Taqman-MGB probe of the present invention is better than the reported quantitative PCR detection method based on SYBR Green I dye, and the detection specificity after application of the probe is better than the quantitative PCR detection based on SYBR Green I dye method.

[0077] 3. Convenient and fast, can be used for on-site testing:

[0078] The isothermal amplification detection primer and detection method provided by the present invention can complete the detection under simple constant temperature conditions (such as a water bath or a metal bath, etc.), and the nucleic acid fluorescent dye SYBR Green or GeneFinder is added to the product. TM Or metal ion indicator dye calcein, etc., can be used to judge the on-site test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Picture 1 .The first set of primers for fluorescent quantitative PCR Ameson- F. Ameson- R and probe Ameson -Probe nucleotide sequence and Ameson The comparison result of SSU rRNA gene of spp.

[0080] Picture 2 .based on Ameson spp .Phylogenetic tree of SSU rRNA gene sequences;

[0081] Picture 3 . Ameson spp. Real-time fluorescence quantitative PCR amplification curve, where 1-10: 4.58×10 9 ~4.58×10 0 copies / μL, 11: negative control;

[0082] Picture 4 . Ameson spp. Real-time fluorescence quantitative PCR standard curve;

[0083] Picture 5 . Ameson spp. plasmid standard as template for nested PCR amplification sensitivity test, where 1-6: round 1 products, templates are 4.58×10 5 ~4.58×10 0 copies / μL; 7-12: second-round PCR products corresponding to products 1-6 as templates; N1 and N2: negative controls;

[0084] Picture 6 . Ameson spp. Nested PCR test results of clinical samples, where 1-6: first-round PCR products using clinical samples as templates; 7-12: second-round PCR products; N1 and N2: negative controls;

[0085] Picture 7 . Ameson spp. Real-time fluorescence quantitative PCR specific test results, among which, 1: Sarcospora; 2-4: Ameson sp.; 5-16: other pathogens; 17: negative control. DETAILED DESCRIPTION

[0086] To further illustrate the method and effect of the present invention, the present invention is further described below in conjunction with the examples. Unless otherwise specified, the reagents and materials in the following examples are all commercially available.

[0087] Embodiment 1: The present invention is to Ameson spp. Effective detection and new Ameson The discovery of sp.

[0088] From 2020 to 2023, we continued to conduct crustacean epidemiological surveys in the main breeding areas of swimming crabs in Rizhao and Weifang, Shandong. In the investigation of the pathogen of Sarcospora, we used PCR detection combined with sequencing analysis. In the PCR detection method, based on Ameson portunus A pair of primers Amp_F / Amp_R were designed for the JQX201209 SSU rRNA (KC915038.1) gene. The corresponding PCR product was 916 bp. The primer sequences are as follows:

[0089] Amp_F: 5'-ATGGCGACCAGTTCTAAGGA-3',

[0090] Amp_R: 5'-GATAACGACGGGCGGTGTGT-3'.

[0091] The reaction system is 12.5 μL Premix Ex Taq Mix (TaKaRa, Dalian), 0.5 μL of 10 μmol / L primer Amp_F / Amp_R, 1 μL of DNA template, and sterile enzyme-free water to 25 μL. The PCR reaction conditions were 95°C pre-denaturation for 5 min; 95°C 45 s, 47°C 35 s, 72°C 40 s, for a total of 25 cycles; 72°C extension for 5 min.

[0092] Through a large number of PCR tests and product sequencing analysis in crustacean epidemiological studies, the following findings were made:

[0093] (1) The primers and probes of the present invention can be used for Ameson spp. Effective detection and Ameson A new species of sp. was discovered.

[0094] right Ameson spp. SSU rRNA gene comparison analysis revealed: Ameson The different species of the genus, including the confirmed and unconfirmed species currently published in NCBI, as well as the different crab sources we have newly discovered Ameson sp. (not yet published). Some of our analysis results are as follows: The microsporidia in the 26 tested swimming crabs were similar to A. portunus (KC915038.1) were 100% similar; the 10 short-bodied big-eyed crab samples tested were also A. portunus (KC915038.1) is 100% similar, and the other 16 short-bodied big-eyed crabs are A. portunus (KC915038.1) and A. pulvis (KC465966.1) has a similarity of 98.5%-99.8%; the 11 red-line dawn crabs, 1 Japanese big-eyed crab and 18 bean-shaped fist crabs tested are all A. pulvis (KC465966.1), with genetic similarities of 99.1%-99.8%, 99.6% and 99.6%-99.8% respectively.

[0095] The nucleotide sequences of the first set of primers and probes of the present invention are Ameson The comparison results of SSU rRNA genes of spp. (MEGA 7.0 software, the same below) are shown in Picture 1 , whose nucleotide sequence is as follows:

[0096] 1) Primer 1 Ameson- F: 5'-ATTTGGTGTCAGCTAATGGTATGG-3',

[0097] 2) Primer 2 Ameson- R: 5'-TTGTCAGTGCTTTCGCCTTTG-3',

[0098] 3) Probe Ameson -Probe:5'–TGAAGTCTGAGGATCCA-3'.

[0099] We have obtained some information on the SSU rRNA gene from epidemiological surveys Ameson sp. sequencing results, and the phylogenetic tree was constructed using MEGA 7.0 software based on the neighbor-joining method (NJ, Neighbor-Joining) (bootstrap 1000 times) Picture 2 The results show that there are four Ameson sp. new species were discovered, such as strains numbered 20210629023, 20211025041, 20211017016 and 20210629017. The above results show that the primers and probes provided by the present invention can be used for the detection of Ameson spp. Effective detection and new Ameson The discovery of sp.

[0100] (2) Existing methods have the problem of low detection specificity.

[0101] The reported SYBR Green I quantitative PCR and nested PCR for myocytosis have the problem of low specificity in detecting the pathogen. In addition, the second round of PCR detection of nested PCR also has the problem of non-specific amplification that affects the judgment of the detection results (see Example 6).

[0102] Embodiment 2: Ameson spp. pMD18- Ameson Construction of recombinant plasmid standards

[0103] Using the primers Amp_F / Amp_R and reaction procedure of Example 1, the infection A. portunus The genomic DNA of swimming crab tissue was used as a template for PCR amplification. The 916 bp PCR product was purified by gel recovery and ligated to the pMD18-T vector (TaKaRa, Dalian, China) and then transformed into E. coli DH5α, the bacterial culture containing the target plasmid was spread on the LB solid plate containing Amp, cultured at 37°C overnight, and a single colony was picked for colony PCR verification using M13 primers. The positive strains were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing verification. Ameson After the bacterial suspension was cultured at 37°C overnight, pMD18- Ameson The plasmid was purified by gel recovery and the concentration was determined by NanoDrop 2000c, the copy number of the target gene was calculated, and the plasmid was stored at -20°C for future use.

[0104] Embodiment 3: Ameson Primers and probes for fluorescent quantitative PCR of spp.

[0105] According to NCBI Ameson spp. SSU rRNA gene sequence, as well as the results of sequencing in recent epidemiological surveys (Example 1), were used to design primers and probes for its conservative sequence. The 5' end of the probe was modified with a fluorescent reporter group 6-FAM, and the 3' end was modified with MGB-NFQ. Both the primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0106] The nucleotide sequences of the designed 4 sets of fluorescence quantitative PCR detection primers and probes are as follows:

[0107] The first set

[0108] 1) Primer 1 Ameson- F: 5'-ATTTGGTGTCAGCTAATGGTATGG-3',

[0109] 2) Primer 2 Ameson-R: 5'-TTGTCAGTGCTTTCGCCTTTG-3',

[0110] 3) Probe Ameson -Probe:5'–TGAAGTCTGAGGATCCA-3'.

[0111] Second set

[0112] 1) Primer 1 Ameson- F: 5'-ATTTGACTCAACGCGGGAAA-3',

[0113] 2) Primer 2 Ameson- R: 5'-GCCATGCACCACCATTTTAC-3',

[0114] 3) Probe Ameson -Probe:5'-ACCAAGCTTATTTATTCAACG-3'.

[0115] The third set

[0116] 1) Primer 1 Ameson- F: 5'-GTTGCGGTTAAAACGTCCGT-3',

[0117] 2) Primer 2 Ameson- R: 5'-TTTCCAGCTTGCTACTGCCC-3',

[0118] 3) Probe Ameson -Probe: 5'-CTGACCTGTAATCTATTTGG-3'.

[0119] The fourth set

[0120] 1) Primer 1 Ameson- F: 5'-ACTGGAGGGAAAGTCTGGTG-3',

[0121] 2) Primer 2 Ameson- R: 5'-CGCGACTACGGACGTTTTAA-3',

[0122] 3) Probe Ameson -Probe: 5'-CAGCAGCCGCGGTA-3'.

[0123] The 5' end of the above probes can also be modified with other fluorescent reporter groups such as TET, JOE, VIC, NED, CY3 and HEX, and the 3' end can also be modified with fluorescent quencher groups such as TAMRA and BHQ. When the negative and positive controls are normal, the sample is considered to have an amplification curve. Amesonspp. test positive, the pathogen load in the sample can also be calculated based on the linear relationship between the template amount and the CT value and the CT value of the test sample.

[0124] The above quantitative detection can also be performed by using the dye method without adding a probe, and adding nucleic acid dyes such as SYBRGreen Ⅰ to the detection system. When the negative and positive detections are normal, the product dissolution curve is a single peak graph, which is considered as the sample Ameson spp. test is positive. This test can also refer to the quantitative PCR method of the probe method to quantify the pathogen load in the sample.

[0125] Example 4: Optimization of reaction conditions for Taqman-MGB fluorescence quantitative PCR

[0126] The first set of primers for quantitative PCR Ameson- F. Ameson- R and probe Ameson -Probe for testing. Premix Ex Taq (Probe qPCR) kit (TakaRa, Dalian, China), the primers in the system Ameson The final concentrations of -F / -R were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 μmol·L -1 , probe Ameson The final concentrations of -Probe were 0.1, 0.2, 0.3, 0.4, and 0.5 μmol·L -1 , 0.2μL ROX Reference Dye II (50×), 1μL DNA template, and ddH2O to make up to 20μL. The reaction temperatures to be optimized were set to 58, 59, 60, 61, 62, and 63℃, respectively. The initial amplification program was 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; reaction temperature 60℃ for 30s, 40 cycles. The detection effect was evaluated by detecting CT value and fluorescence intensity increase value (ΔRn). Ameson The final concentration of -F / -R was 0.2 μmol·L -1 , probe Ameson -Probe final concentration was 0.3 μmol·L -1 The reaction conditions were 95°C for 30s, 95°C for 5s, and 60°C for 30s, for a total of 40 cycles. The optimized system and reaction temperature were used in the experiments of the subsequent examples, hereinafter referred to as the first set of Taqman-MGB qPCR methods.

[0127] Example 5: Standard curve, detection sensitivity and repeatability of Taqman-MGB fluorescence quantitative PCR

[0128] (1) Standard curve and detection sensitivity

[0129] pMD18- Ameson The recombinant plasmid was diluted 10-fold using EASY Dilution (for Real Time PCR) to a concentration of 4.58 × 10 9 -4.58×10 0 The plasmid standard with 100 copies / μL was used as a template and the first set of Taqman-MGB qPCR method was used for amplification. The results showed that the detection sensitivity of qPCR within 40 cycles was 4.58 copies / reaction ( Picture 3 ), the linear expression of the logarithmic value (x) of nucleic acid copy number and CT value (y) is y=-3.119x+38.978, R 2= 0.998, E=109.201%( Picture 4 ). The detection sensitivity of this method is better than the detection sensitivity of 20 copies / reaction of the SYBR Green I real-time quantitative PCR detection method of Sarcocystis by Chen Tiantian et al. (2017), and is more suitable for early diagnosis and early warning of pathogen infection.

[0130] (2) Repeatability of intra-batch and inter-batch testing

[0131] The concentration is 4.58×10 9 ~4.58×10 0 copies / μL pMD18- Ameson The recombinant plasmid was used as a template for intra-batch and inter-batch repeated tests, with 3 parallel tests for each concentration. The intra-batch and inter-batch coefficients of variation were 0.06%-1.37% and 0.44%-2.13%, respectively (Table 1), indicating that the quantitative PCR method based on the primers and probes of the present invention has good stability in detection within this template concentration range.

[0132] Table 1 Ameson spp. Fluorescence quantitative PCR repeatability test results

[0133]

[0134] Example 6: Comparison of Taqman-MGB fluorescence quantitative PCR detection method and nested PCR

[0135] pMD18- Ameson The tissue nucleic acid of the clinical sample containing the plasmid was used as a template to perform the first set of Taqman-MGB qPCR method of the present invention and the nested PCR method (diagnostic procedures for myosporosis of Portunus trituberculatus, aquatic industry standard, SC / T 7239-2020) for detection and comparison. AmesonThe plasmid contains the sequence corresponding to the first round of primers in this nested format. In the detection of plasmid standard as template, the detection sensitivity of Taqman-MGB qPCR test within 40 cycles was 4.58 copies / reaction; in the nested PCR detection, the detection sensitivity of the first and second rounds was 4.58×10 3 copies / reaction and 4.58 copies / reaction, but obvious nonspecific amplification occurred in the second round of nested PCR ( Picture 5 In the tissue nucleic acid detection of clinical samples, this non-specific amplification also exists in the second round of nested PCR detection ( Picture 6 ), this non-specific amplification is not conducive to the accurate judgment of the test results. In terms of detection time, the quantitative PCR method is much shorter than the nested PCR method, and the cover is not opened during the detection process, which can effectively reduce the occurrence of contamination in molecular detection.

[0136] Example 7: Specificity evaluation of Taqman-MGB fluorescence quantitative PCR detection method

[0137] In the first set of Taqman-MGB qPCR assays, pMD18- Ameson The plasmid nucleic acid was used as the positive control, the healthy swimming crab nucleic acid was used as the template as the negative control, and ddH2O was used as the template as the blank control. Other pathogenic nucleic acids are shown in Table 2. The experiment was repeated three times. The test results showed that this method can specifically detect crab tissue Ameson spp. (the positive has been confirmed by sequencing), and has no cross-reaction with DNA from other common shrimp and crab pathogens ( Picture 7 ).

[0138] Table 2 List of pathogens used in specific detection

[0139]

[0140] Example 8: Clinical sample testing

[0141] The qPCR method of the present invention can be used for Ameson Detection of clinical samples for Microsporidia sp.

[0142] The first set of Taqman-MGB qPCR method and conventional PCR detection method of the present invention were used to detect hemolymph nucleic acid of 40 clinical samples of swimming crabs collected from a breeding farm in Rizhao, Shandong in 2022. The results showed that 33 positive samples were detected by qPCR, with a detection rate of 82.5%, and the CT value was between 12.26-35.36, which was converted into a copy number of 4.33×10 0 -1.11×10 8copies / μL; conventional PCR (one round of PCR in the nested PCR method in the aquatic products industry standard SC / T7239-2020) detected 11 positive samples with a detection rate of 27.5% (Table 3).

[0143] The first set of Taqman-MGB qPCR method of the present invention detected the presence of 549697 genes in all tissues of 6 swimming crabs suspected of having "toothpaste disease" collected from a farm in Weifang, Shandong in 2023. Ameson spp. Microsporidia were positive, with a pathogen load of 9.23×10 0 -1.46×10 9 copies / mg. The pathogen was confirmed to be Sarcospora by PCR product sequencing analysis. The pathogen load of two swimming crabs numbered 20231125002 and 20231125006 was as high as 1.63×10 7 -1.46×10 9 copies / mg, and the myosporidial loads in the pliers and abdominal muscles were higher than those in the hepatopancreas, myocardium, and gill tissues (Table 4).

[0144] Table 3 Ameson Comparison of spp.qPCR and conventional PCR in the detection of clinical samples

[0145]

[0146] Table 4 Comparison of conventional PCR and qPCR for the detection of clinical samples of Sarcocystis

[0147]

[0148] Embodiment 9: Ameson Isothermal amplification primers and their applications for spp.

[0149] According to the published Ameson sp. is different from what we found in our epidemiological survey. Ameson sp., using online software ( http: / / primerexplorer.jp / e / ), designed for Ameson spp. The four sets of primers for isothermal amplification are as follows:

[0150] First set:

[0151] 1) Primer 1 Ameson -F3:5'-CGGTAATACCAGCTCCAGGA-3',

[0152] 2) Primer 2 Ameson -B3:5'-GCCTCTGTCCTTGATCAACA-3',

[0153] 3) Primer 3 Ameson -FIP:

[0154] 5'-TCCAGCTTGCTACTGCCCCTATTTTTTTAAAACGTCCGTAGTCGCG-3',

[0155] 4) Primer 4 Ameson -BIP:

[0156] 5'-AGCTAATGGTATGGGGAGGGGTTTTTGCTTTCGCCTTTGCTCCTC-3',

[0157] 5) Primer 5 Ameson -LF:5'-AAATAGATTACAGGTCAGTCCCAAG-3'.

[0158] Second set:

[0159] 1) Primer 1 Ameson -F3:5'-GCTTGGGACTGACCTGTA-3',

[0160] 2) Primer 2 Ameson -B3:5'-CGTATTCGATCCTCTAGCCT-3',

[0161] 3) Primer 3 Ameson -FIP:

[0162] 5'-CATACCATTAGCTGACACCAAATTGTTTTGTCAACAGATAGATAGGGGC-3',

[0163] 4) Primer 4 Ameson -BIP:

[0164] 5'-GAGGGGTGAAGTCTGAGGATTTTTTTGATCAACAGAATCAATCTTTGTC-3',

[0165] 5) Primer 5 Ameson -LF:5'-TCTTTTCCAGCTTGCTACT-3',

[0166] 6) Primer 6 Ameson -LB:5'-CCATGCAGGAGGAGCA-3'.

[0167] The third set:

[0168] 1) Primer 1 Ameson-F3:5'-GATCGAATACGATTAGATACCG-3',

[0169] 2) Primer 2 Ameson -B3:5'-TTGGTAAGTTTTCCCGCG-3',

[0170] 3) Primer 3 Ameson -FIP:

[0171] 5'-TCCCCCCGGAGATCTATTTTGATTTTTTAGTAGTTCTAGCAGTGACCG-3',

[0172] 4) Primer 4 Ameson -BIP:

[0173] 5'-GTACATGCGCAAGCAAGAAACTTTTTTGAGTCAAATTAAGCCGAAC-3',

[0174] 5) Primer 5 Ameson -LB:5'-AAAGAAATTGACGGAAGACTACCAC-3'.

[0175] The fourth set:

[0176] 1) Primer 1 Ameson -F3:5'-GTAAATGGTGGTGCATGG-3',

[0177] 2) Primer 2 Ameson -B3:5'-TGTTCATGAGTGCTTTCAG-3',

[0178] 3) Primer 3 Ameson -FIP:

[0179] 5'-TCTTTCGAACAAGCTATTCTGAGATTTTTCAGATGGAGTGATTTTGTGAT-3',

[0180] 4) Primer 4 Ameson -BIP:

[0181] 5'-TGCACGCGCAATACAATGTTTTTTCCCTCAATCCCTCATGTT-3'.

[0182] Embodiment 10: Ameson Application of isothermal amplification detection of spp.

[0183] The reaction system of the above primers based on LAMP detection is calculated in 25 μL, including the following components: 10×Isothermalamplification buffer 2.5 μL, MgSO4 with a final concentration of 4.0-12.0 mM, Betaine with a final concentration of 0.1-1.4 mM, dNTPs with a final concentration of 0.8-1.8 mM, 20 μM Ameson -FIP / Ameson -BIP 2 μL, 10 μM Ameson -F3 / Ameson -B3 0.5 μL, 20 μM Ameson -LF / Ameson -LB 1 μL, final concentration 0.128-0.576U / uL Bst 2.0WarmStart ® DNA polymerase (8000U / mL, New England BioLabs), Ameson spp. DNA template 1μL, and add water to 25μL, in addition, 0.5-2.5μM 20×EvaGreen can be added to the system ® Dye (Biotium) 1 μL. The reaction temperature is 60.0℃-68.0℃, placed in a real-time fluorescence quantitative PCR instrument, and reacted for 60 minutes (60 cycles, 1 minute each cycle).

[0184] In the LAMP assay of the above-mentioned primer sets, the following primers can be used: Ameson -F3, Ameson -B3, Ameson -FIP and Ameson -BIP; can also be used Ameson -F3, Ameson -B3, Ameson -FIP, Ameson -BIP and Ameson -LF; you can also use Ameson -F3, Ameson -B3, Ameson -FIP, Ameson -BIP and Ameson -LB; you can also use Ameson -F3, Ameson -B3, Ameson -FIP, Ameson -BIP, Ameson -LF and Ameson -LB.

[0185] In the above LAMP assay, EvaGreen was added ®The dye system is placed in a quantitative PCR instrument. The detection reaction system and temperature can be optimized based on the low CT value and reagent cost in the amplification. In this test, the linear relationship between the template amount and the CT value can be used to determine the amount of DNA in the test sample. Ameson sp. loading for quantitative analysis; no EvaGreen added ® When dyeing, the reaction conditions can be optimized based on the bright bands of the product ladder electrophoresis bands and the reagent cost. In this test, 2% volume of nucleic acid dye (such as GeneFinder TM ), on-site diagnosis of samples under simple isothermal conditions is performed through color changes in the product tube.

[0186] The primers and probes of the present invention can be used for non-disease diagnosis and treatment purposes. Ameson spp., and can also be used for the development of detection reagents or kit products, and for new Ameson The discovery of sp.

Claims

1. A crustacean Ameson spp. Microsporidia detection primers and probes, characterized in that: The nucleotide sequences of the fluorescent quantitative PCR detection primers and probes are as follows: 1) Primer 1 Ameson- F: 5'-ATTTGGTGTCAGCTAATGGTATGG-3', 2) Primer 2 Ameson- R: 5'-TTGTCAGTGCTTTCGCCTTTG-3', 3) Probe Ameson -Probe: 5' -TGAAGTCTGAGGATCCA-3'.

2. A crustacean according to claim 1 Ameson The application of primers and probes for detecting microsporidia spp. is characterized by: For non-disease diagnosis and treatment purposes Ameson Detection and new Ameson The discovery of sp.

3. The crustacean according to claim 2 Ameson The application of primers and probes for detecting microsporidia spp. is characterized by: The 5' end of the probe is modified with a fluorescent reporter group 6-FAM, and the 3' end is modified with a fluorescent quencher group MGB-NFQ.

4. The crustacean according to claim 2 Ameson The application of primers and probes for detecting microsporidia spp. is characterized by: The final concentrations of primers and probes in the detection system are both 0.1-0.5 μM.

5. The crustacean according to claim 2 Ameson The application of primers and probes for detecting microsporidia spp. is characterized by: When using fluorescent quantitative PCR detection primers and probes 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=-3.119x+38.

978.

6. The crustacean according to claim 2 Ameson The application of primers and probes for detecting microsporidia spp. is characterized by: Including products used to prepare detection reagents or test kits.

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