Detection nucleotide sequence of eriocheir sinensis spiroplasma and application thereof

Abstract

The application belongs to the technical field of microorganism detection, and particularly relates to a detection nucleotide sequence of a Chinese mitten crab spiroplasma and application thereof. The application designs a fluorescence quantitative PCR detection primer and probe based on a probe method for the ftsZ gene of the Chinese mitten crab spiroplasma. The detection sensitivity of the target gene in the detection is 3.3 copies / reaction, and the method has the advantages of specificity, rapidness and stable results, and is suitable for early monitoring and epidemiological investigation of river crab spiroplasma disease.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to the detection of nucleotide sequences of *Eriocheir sinensis* and their applications. Background Technology

[0002] The Chinese mitten crab (Eriocheir sinensis), also known as the river crab or mitten crab, is one of the important farmed freshwater crustaceans in my country. Spiroplasma disease in Chinese mitten crabs is currently a significant disease, also known as "trembling disease" because infected crabs often exhibit trembling appendages in the later stages of the disease. This disease is caused by Spiroplasma eriocheiris and is widespread in crab farming areas, with morbidity rates reaching 30%-70% in some areas, and mortality rates exceeding 80% in severe cases. In recent years, reports of spiroplasma infections have also been reported in other species such as the red swamp crayfish (Procambarus clarkii), Litopenaeus vannamei, Macrobrachium rosenbergii, and M. nipponensis. Given the serious harm caused by spiroplasma disease in Chinese mitten crabs, it has been classified as a Class III animal disease by the Ministry of Agriculture and Rural Affairs.

[0003] Sensitive and specific diagnosis of pathogens is the foundation of effective pathogen control. Currently, most detection methods for *Eriocheir sinensis* are based on immunology and molecular biology, including colloidal gold immunochromatographic assay (GICA), fluorescence in situ hybridization (FISH), PCR, nested PCR, multiplex PCR, quantitative real-time PCR, multienzyme isothermal rapid amplification (MIRA), and MIRA-Lateral Flow Dipstick (LFD). However, these methods still have many problems, such as low detection sensitivity, weak conservation of target gene sequences, cumbersome operation, long detection time, and low specificity. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a detection nucleotide sequence for *Eriocheir sinensis*. Targeting the *Eriocheir sinensis* ftsZ gene, this invention designs a fluorescence quantitative PCR detection nucleotide sequence based on a novel Taqman-MGB probe, and also provides an application method for the detection nucleotide sequence. This method offers the advantages of sensitive, specific, rapid, and quantitative analysis.

[0005] The detection nucleotide sequence of *Eriocheir sinensis* snails described in this invention includes four sets of real-time PCR detection primers and probes, as follows:

[0006] (1) Nucleotide sequences of primers and probes for the first set of real-time PCR detection:

[0007] 1) Primer 1: S.e_F: 5'-GGAGGCGTACCGTTAAAAGATTC-3', as shown in SEQ ID NO: 1;

[0008] 2) Primer 2: S.e_R: 5'-AGGAACGGCAATTAAATCAGTGAT-3', as shown in SEQ ID NO: 2;

[0009] 3) Probe: S.e_P: 5'-CTGCGCCAAGGTG-3', as shown in SEQ ID NO: 3;

[0010] (2) Nucleotide sequences of primers and probes for the second set of real-time PCR detection:

[0011] 1) Primer 1: S.e_F: 5'-TCTTAAAAGGAGCCGACATGGT-3', as shown in SEQ ID NO: 4;

[0012] 2) Primer 2: S.e_R: 5'-CACGGGCAATTTTTGCAATA-3', as shown in SEQ ID NO: 5;

[0013] 3) Probe: S.e_P: 5'-TGTGGCCGCCGGGA-3', as shown in SEQ ID NO: 6;

[0014] (3) Nucleotide sequences of primers and probes for the third set of real-time PCR detection:

[0015] 1) Primer 1: S.e_F: 5'-CCATTCAGGGAACCGAAGAA-3', as shown in SEQ ID NO: 7;

[0016] 2) Primer 2: S.e_R: 5'-TAACGGTACGCCTCCAATTAC-3', as shown in SEQ ID NO: 8;

[0017] 3) Probe: S.e_P: 5'-CTGCGCCAACATGTTGATTCGTTAAT-3', as shown in SEQ ID NO: 9;

[0018] (4) Nucleotide sequences of primers and probes for the fourth set of real-time PCR detection:

[0019] 1) Primer 1: S.e_F: 5'-GCGATTGAAGCAGCCAATAAA-3', as shown in SEQ ID NO: 10;

[0020] 2) Primer 2: S.e_R: 5'-CCCGTTACGTTAATAATGGCATC-3', as shown in SEQ ID NO: 11;

[0021] 3) Probe: S.e_P: 5'-TTTCGCACCGCGAATTGAAGCTTC-3', as shown in SEQ ID NO: 12.

[0022] The target sequence for detecting the nucleotide sequence of *Eriocheir sinensis* is located in the ftsZ gene nucleotide sequence of *Eriocheir sinensis*.

[0023] The detection nucleotides of *Eriocheir sinensis* described in this invention can be used for the detection and quantitative analysis of *Eriocheir sinensis* for non-disease diagnostic and therapeutic purposes, including for the preparation of detection reagents or kits.

[0024] When performing the test, you can choose to use both primers and probes or just primers.

[0025] Preferably, when using probes, the first and second sets of quantitative PCR primers and probes use Taqman-MGB probes with a 5' end modified with a 6-carboxyfluorescein (6-FAM) fluorescent reporter group and a 3' end modified with a MGB (Minor Groove Binder)-NFQ (Nonfluorescent Quencher) fluorescent quencher group; the third and fourth sets of quantitative PCR primers and probes use Taqman probes with a 5' end modified with a fluorescent reporter group and a 3' end modified with a fluorescent quencher group. When probes are not used, a chimeric nucleic acid fluorescent dye that can bind to double-stranded DNA and emit fluorescence should also be added to the detection system.

[0026] In the real-time PCR detection system, the final concentrations of primers and probes are 0.1-0.5 μmol / L and 0.1-0.5 μmol / L, respectively. Preferably, the final concentrations of primers and probes for the first set of real-time PCR detection are both 0.3 μmol / L.

[0027] The detection procedure for quantitative real-time PCR is as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; 58-63℃ (preferably 60℃) for 30s, 40 cycles.

[0028] Analysis of quantitative real-time PCR (qPCR) results: Positive samples show an amplification curve; for samples without probes, the melting curve should show a single peak. In pathogen quantification, the pathogen load in the sample is calculated based on the linear relationship between the logarithm of the target gene copy number (x) in the plasmid standard and its threshold cycle number (Ct) (y), as well as the Ct value of the actual tested sample. The linear equation for the first set of qPCR primers and probes in qPCR detection based on the Taqman-MGB probe is y = -3.18x + 40.528.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention exhibits high sensitivity, with a detection sensitivity of 3.3 copies / reaction for the target gene of *Eriocheir sinensis*, which is superior to other reported detection methods for this pathogen. It also demonstrates rapid detection, completing the test within 1 hour, and high specificity, showing no cross-reaction with 15 other aquatic animal pathogens during the detection process. Attached Figure Description

[0031] Figure 1 The first set of primers and probe nucleotide sequences for detecting *Eriocheir sinensis* snails;

[0032] Figure 2 A real-time PCR amplification curve using plasmid standards from the Chinese mitten crab snail as templates;

[0033] Figure 3 Standard curve of TaqMan-MGB real-time PCR for *Eriocheir sinensis* snail protozoa;

[0034] Figure 4 Amplification curves of TaqMan-MGB real-time PCR for the specific detection of *Eriocheir sinensis* (I);

[0035] Figure 5 Amplification curves for specific detection of *Eriocheir sinensis* by TaqMan-MGB real-time PCR (II). Detailed Implementation

[0036] To further illustrate the method and effects of the present invention, the following embodiments are provided for further explanation. Unless otherwise specified, all pharmaceuticals and materials used in the following embodiments are commercially available.

[0037] Example 1: Nucleotide sequence design for detection of *Eriocheir sinensis* snails

[0038] Primers S.e_ftsZ_F: 5'-AGCAGGAGTGCAGGGAGT-3' (SEQ ID NO: 13) and S.e_ftsZ_R: 5'-GTTGGCGTCGTTTAGGGT-3' (SEQ ID NO: 14) were designed to target the ftsZ gene of *Eriocheir sinensis*. PCR amplification was performed using 2×Accurate Taq premix (Hunan Aike Rui Biotechnology Co., Ltd.) (product: 730 bp). The PCR reaction program was: 95℃ 5 min; [95℃ 30 s, 58℃ 30 s, 72℃ 40 s]×35; 72℃ 8 min. Twenty-three strains (Table 1) were collected, amplified by PCR, and sequenced. The results were compared with the ftsZ gene sequences of three *Eriocheir sinensis* strains currently published by NCBI (National Center for Biotechnology Information). Based on the determined conserved nucleotide sequences, four sets of fluorescent quantitative PCR detection primers and probes of this invention were designed.

[0039] Table 1. Nucleic acid samples from *Eriocheir sinensis* snails.

[0040]

[0041] Specifically, the nucleotide sequences of the first set of primers and probes designed for real-time PCR detection (TaqMan-MGB probe) are as follows:

[0042] 1) Primer 1: S.e_F: 5'-GGAGGCGTACCGTTAAAAGATTC-3',

[0043] 2) Primer 2: S.e_R: 5'-AGGAACGGCAATTAAATCAGTGAT-3',

[0044] 3) Probe: S.e_P: 5'-CTGCGCCAAGGTG-3'.

[0045] The nucleotide sequence information of the primers and probes for the first set of real-time PCR detection in the ftsZ gene is shown in [link to relevant documentation]. Figure 1 , Figure 1 In the diagram, yellow represents A, red represents T, blue represents C, and green represents G. (From...) Figure 1 It is evident that the designed primer and probe nucleotide sequences are completely identical to the nucleotide sequences of the above 26 strains (23 collected strains + 3 strains published by NCBI) of *Eriocheir sinensis* snails.

[0046] The following examples use the first set of detection primers and probes as an example to illustrate their detection applications.

[0047] Example 2: Construction of recombinant plasmid standard pMD18_S.e

[0048] In Example 1, the 730bp PCR product obtained from PCR amplification using the S.e_ftsZ_F / S.e_ftsZ_R primers was purified via gel extraction, ligated into the pMD18-T vector (TaKaRa, Dalian), and then transformed into Escherichia coli DH5α competent cells (TaKaRa, Dalian). The cells were plated on LB agar (containing 100 μg / mL ampicillin). Plasmids containing the recombinant plasmid pMD18_S.e were extracted from positive strains using a column-based plasmid extraction kit (Tiangen Biotech, Beijing). The concentration was determined using a NanoDrop 2000c Spectrophotometer (ThermoScientific, USA). The formula for calculating the target gene copy number is as follows: Copy number (copies / μL) = 6.02 × 10⁻⁶. 23 ×[Plasmid standard concentration (ng / μL) × 10 -9 / (number of base pairs in the ligation vector + number of base pairs in the amplified fragment) × 660]. Then, perform a 10-fold serial dilution using EASY Dilution (for Real Time PCR) (TaKaRa, Dalian) to obtain 3.3 × 10⁻⁶. 9 -3.3×10 0 Copies / μL of pMD18_S.e. should be stored at -20℃ for later use.

[0049] Example 3: Optimization of detection primer and probe concentrations

[0050] Quantitative detection was performed using Premix Ex Taq. TMThe Probe qPCR kit (TakaRa, Dalian) was used. Primers S.e_F / S.e_R were added at final concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 μmol / L, and probe S.e_P was added at final concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 μmol / L. 10 μL of Premix Ex Taq (Probe qPCR) (2×), 0.2 μL of ROX ReferenceDye II (50×), and 1 μL of DNA template were added, with the final volume brought to 20 μL using ddH2O. The assay was performed using an Applied Biosystems QuantStudio 3 (Thermo Fisher Scientific, USA). The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ for 30 s, for 40 cycles. Considering factors such as Ct value, fluorescence intensity increase (ΔRn), and reagent cost, the appropriate concentrations of primers and probes were determined to be 0.3 μmol / L and 0.3 μmol / L, respectively. The results were used in subsequent detections using this method.

[0051] Example 4: Sensitivity and Repeatability Evaluation of Real-Time PCR Detection

[0052] (1) Detection sensitivity

[0053] 3.3 × 10⁻⁶ obtained by a 10-fold serial dilution 9 -3.3×10 0 Using pMD18_S.e copies / μL as a template, quantitative real-time PCR was performed, and the results are as follows: Figure 2 This indicates that its detection sensitivity is 3.3 copies / reaction over 40 detection cycles. Figure 2 In the middle, 1-10: the corresponding template concentrations are 3.3×10⁻⁶. 9 3.3×10 8 3.3×10 7 3.3×10 6 3.3×10 5 3.3×10 4 3.3×10 3 3.3×10 2 3.3×10 1 and 3.3×10 0 pMD18_S.e. copies / μL; N: negative control.

[0054] Correlation analysis was performed on the logarithm (x) of the target gene copy number in pMD18_S.e and its corresponding threshold cycle number Ct (y), yielding the standard curve: y = -3.18x + 40.528, R0. 2=0.997, amplification efficiency of 106.3% ( Figure 3 ).

[0055] (2) Repeatability assessment

[0056] With 3.3×10 9 -3.3×10 0 Using pMD18_S.e plasmid standard (copies / μL) as templates, three replicates were set up for each gradient for intra-batch and inter-batch detection. The mean Ct, standard deviation, and coefficient of variation were calculated. The results showed that the TaqMan-MGB probe-based quantitative PCR detection method of this invention has good reproducibility, with intra-batch and inter-batch coefficients of variation ranging from 0.03% to 1.71% and 0.38% to 1.37%, respectively (Table 2).

[0057] Table 2 Repeatability of Real-Time PCR Detection

[0058]

[0059] Example 5: Detection of Specificity by Real-Time PCR

[0060] Specificity testing was performed in two batches. Using DNA from each pathogen or positive sample in Table 3 as templates and sterile, enzyme-free water as a negative control, real-time quantitative PCR was performed on a Bio-Rad CFX Opus 96 Real-Time PCR System. Using DNA from each pathogen or positive sample in Table 4 as templates, and pMD18_S.e and sterile, enzyme-free water as positive and negative controls respectively, specificity testing was performed on an Applied Biosystems QuantStudio 3. Each test was repeated three times. The results of the three tests are shown below. Figure 4 and Figure 5 The results showed that the fluorescence quantitative PCR method of the present invention can specifically detect *Eriocheir sinensis* and has no cross-reaction with 15 other common pathogens in fish and crustaceans. Figure 4 In the amplification curves, the pathogen nucleic acid templates corresponding to numbers 1-11 are shown in Table 3, and N is the negative control. Figure 5 In the amplification curves, the pathogen nucleic acid templates corresponding to numbers 1-11 are shown in Table 4. P: positive control (pMD18_S.e), N: negative control.

[0061] Table 3. Pathogens used for specific detection of *Eriocheir sinensis* in Chinese mitten crab (Part 1)

[0062]

[0063] Table 4. Pathogens used for specific detection of *Eriocheir sinensis* in Chinese mitten crab (Part II)

[0064]

[0065] Twenty samples collected from Tianjin, Shandong, and Jiangsu provinces were analyzed for DNA extraction using a centrifuged column-type marine animal tissue genomic DNA extraction kit (Tiangen Biotech, Beijing). TaqMan-MGB real-time PCR was then performed, and the pathogen load was calculated as: pathogen copy number (copies / μL) × total extracted DNA volume (μL) / sample mass (mg). Quantitative analysis showed that the viral load of *Eriocheir sinensis* in the tested tissue samples ranged from 1.3 × 10⁻⁶. 1 -3.8×10 7 copies / mg (Table 5).

[0066] Table 5. Virus load of *Eriocheir sinensis* in clinical samples.

[0067]

Claims

1. A primer and probe for detecting the mitten crab snail *Eriocheir sinensis*, characterized in that: The kit includes two primers and one probe for quantitative real-time PCR detection, with the following nucleotide sequences: 1) Primer 1: S.e_F: 5'-GGAGGCGTACCGTTAAAAGATTC-3', 2) Primer 2: S.e_R: 5'-AGGAACGGCAATTAAATCAGTGAT-3', 3) Probe: S.e_P: 5'-CTGCGCCAAGGTG-3'.

2. The detection primers and probes for the mitten crab snail protozoan as described in claim 1, characterized in that: The target sequences of the detection primers and probes are located in the nucleotide sequence of the ftsZ gene of the Chinese mitten crab snail protozoan.

3. The application of the detection primers and probes for *Eriocheir sinensis* as described in claim 1 in the detection and quantitative analysis of *Eriocheir sinensis* for purposes other than disease diagnosis and treatment.

4. The application as described in claim 3, characterized in that: When performing the test, primers and probes can be used together or primers only.

5. The application as described in claim 4, characterized in that: The probe is modified with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

6. The application as described in claim 5, characterized in that: The probe is a Taqman-MGB probe, with 6-FAM modified at the 5' end and MGB-NFQ modified at the 3' end.

7. The application as described in claim 3, characterized in that: This includes products used to prepare diagnostic reagents or kits.

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