Primer group for specifically detecting tortoise herpesvirus type I in animals and application of primer group in preparation of kit
By designing specific primer sets and using real-time quantitative PCR technology, the problem of rapid and accurate detection of turtle herpesvirus type I was solved, achieving high sensitivity and specificity in detection, helping farmers to detect diseases in a timely manner and reduce losses.
Patent Information
- Application Number
- CN202511454418.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-13
- Publication Date
- 2025-12-09
AI Technical Summary
The lack of a rapid and accurate method for detecting whether farmed turtles are infected with turtle herpesvirus type I in the current technology makes it difficult for farmers to detect and deal with the disease in a timely manner, resulting in significant losses.
The primer set designed for the specific detection of turtle herpesvirus type I and its application in the preparation of the kit includes forward primers, reverse primers and fluorescent probes. Combined with real-time quantitative PCR technology, it provides PCR reaction premix, positive control reference and negative control reference to achieve specific and high-sensitivity detection of turtle herpesvirus type I.
It achieves highly sensitive and specific detection of turtle herpesvirus type I, enabling timely detection of infection during the breeding process and reducing losses.
Smart Images
Figure CN121087232A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal disease detection products, and more particularly to turtle herpesvirus type I detection products. Background Technology
[0002] Tortoise herpesvirus type I belongs to the Herpesviridae family and is a highly contagious virus. Early clinical symptoms in infected turtles include lethargy with closed eyes, loss of appetite, and even complete anorexia. As the disease progresses, white mucus begins to secrete from the mouth and nose, and obvious ulcers appear on the oral mucosa. Scraping with a cotton swab reveals white necrotic tissue, and continuous exudation occurs. Subsequently, white discharge also appears from the eyes, gradually covering the entire eyeball. Affected turtles often excrete green, watery feces, and their condition deteriorates rapidly, manifesting as emaciation and weakness, leading to death from exhaustion within a few days. This disease is highly contagious; once one individual becomes infected, almost all turtles in the same tank are susceptible. Due to the lack of suitable technology for rapid and accurate on-site testing in farms to detect turtle herpesvirus infection, breeders often find it difficult to detect abnormalities and take appropriate measures in time. Often, the infection is only discovered after symptoms appear, at which point irreversible and significant losses have already occurred.
[0003] Quantitative real-time PCR (qPCR) is a method that adds fluorescent dyes or probes to conventional PCR techniques. It utilizes the accumulation of fluorescence signals to monitor the entire PCR process in real time and uses a standard curve to quantify templates of unknown concentrations. As an internationally recognized standard method for nucleic acid quantification, qPCR is the preferred method for rapid detection of gene expression levels. It not only represents a leap from qualitative to quantitative PCR but also offers higher sensitivity, stronger specificity, better repeatability, and a higher degree of automation compared to conventional PCR.
[0004] If a method for detecting turtle herpesvirus type I can be developed based on real-time quantitative PCR technology, it will help farmers detect and monitor turtle herpesvirus type I diseases during aquaculture. Summary of the Invention
[0005] The purpose of this invention is to provide a primer set for the specific detection of turtle herpesvirus type I in animals and its application in the preparation of a reagent kit, so as to solve the problem that there is a lack of products in the prior art for the rapid and accurate detection of whether farmed turtles are infected with turtle herpesvirus type I.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A primer set for the specific detection of turtle herpesvirus type I in animals, comprising:
[0008] The forward primer TH-M1-F has the sequence shown in SEQ ID NO: 1;
[0009] The reverse primer TH-M1-R has the sequence shown in SEQ ID NO: 2;
[0010] The fluorescent probe TH-M1-P has the sequence shown in SEQ ID NO: 3;
[0011] The fluorescent probe TH-M1-P is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.
[0012] Furthermore, the fluorescent probe TH-M1-P is labeled with the fluorescent group FAM at its 5' end and the quenching group MGB at its 3' end.
[0013] The present invention also provides the application of a primer set for the specific detection of turtle herpesvirus type I in animals in the preparation of a reagent kit.
[0014] Furthermore, the kit also includes PCR reaction premix, positive control reference, negative control reference and / or ddH2O.
[0015] Furthermore, the positive control reference is a positive plasmid containing the pol sequence of turtle herpesvirus type I, and the negative control reference is ddH2O.
[0016] The present invention also provides a real-time fluorescent PCR reaction system using the primer set for the specific detection of turtle herpesvirus type I in animals, with a total volume of 25 μL, including 12.5 μL of 2× PCR reaction premix, 1 μL of 10 μM forward primer TH-M1-F, 1 μL of 10 μM reverse primer TH-M1-R, 0.5 μL of 10 μM fluorescent probe, 5 μL of DNA template, and 5 μL of ddH2O.
[0017] Further, the 2× reaction premix comprises: Tris-HCl (pH 8.3–8.8) 50 mM, 50mM, 5 mM, dNTPs 200 µM, Taq DNA Polymerase 0.1 U / µL, BSA / glycerol 0.5 mg / mL / 10% v / v, surfactant 0.1%.
[0018] The advantages of this invention are: based on the pol sequence of turtle herpesvirus type I as a target, a primer-probe set capable of specifically detecting turtle herpesvirus type I was designed. The detection sensitivity for positive plasmids containing the pol sequence of turtle herpesvirus type I reaches 1×10⁻⁶. 0The detection method of this invention has strong specificity and repeatability, and can provide farmers with an effective technical product for the detection and monitoring of turtle herpesvirus type I disease during animal breeding. Attached Figure Description
[0019] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:
[0020] Figure 1 This is a diagram showing the amplification results of the two turtle herpesvirus type I primer and probe sets in Example 1;
[0021] Figure 2 This is a diagram showing the specificity test results of Example 2;
[0022] Figure 3 This is a graph showing the sensitivity test results of Example 3;
[0023] Figure 4 This is a graph showing the negative stability test results of Example 4;
[0024] Figure 5 This is a graph showing the test results for turtle herpesvirus type I in 20 actual samples. Detailed Implementation
[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.
[0026] Unless otherwise specified, the reagents and equipment used in this invention are commercially available, and the operating methods are conventional techniques in this technical field.
[0027] Example 1: Design of primer set for detecting turtle herpesvirus type I
[0028] 1 Experimental Methods
[0029] 1.1 Primer Design
[0030] The complete genome sequence of turtle herpesvirus type I was searched in GenBank and compared using BLTHT analysis. Using the pol sequence as the target gene, two sets of specific primers and probes were designed using PrimerExpress primer design software based on the principles of real-time fluorescent PCR primer and probe design.
[0031] Turtle herpesvirus type I primer and probe set TH-M1:
[0032] Forward primer TH-M1-F: 5'- TCTATTGGGCGAGCTGTTGAC-3' (SEQ ID NO: 1);
[0033] Reverse primer TH-M1-R: 5'-TTTTCATGCGTAGGCGTATGG-3' (SEQ ID NO: 2);
[0034] Fluorescent probe TH-M1-P: 5'-CTGGCCTTGCGAAAA-3' (SEQ ID NO: 3);
[0035] The fluorescent probe TH-M1-P is labeled with the fluorescent group FAM at its 5' end and the quencher group MGB at its 3' end.
[0036] Turtle herpesvirus type I primer and probe set TH-M2:
[0037] Forward primer TH-M2-F: 5'-TGGCCACCCGAGATTATATCC-3' (SEQ ID NO: 4);
[0038] Reverse primer TH-M2-R: 5'-GGGAAGTCGGCCAGAAACTT-3' (SEQ ID NO: 5);
[0039] Fluorescent probe TH-M2-P: 5'-CCATTGGAGCGAACG-3' (SEQ ID NO: 6);
[0040] The fluorescent probe TH-M2-P is labeled with the fluorescent group FAM at its 5' end and the quencher group MGB at its 3' end.
[0041] 1.2 Perform PCR reaction
[0042] The real-time fluorescence PCR reaction system is shown in Table 1, with a total volume of 25 μL.
[0043] Table 1 Real-time fluorescence PCR reaction system
[0044] The 2× reaction premix consists of: Tris-HCl (pH 8.3–8.8) 50 mM. 50mM, 5 mM, dNTPs 200 µM, Taq DNA Polymerase 0.1 U / µL, BSA / glycerol 0.5 mg / mL / 10% v / v, surfactant 0.1%.
[0045] The real-time fluorescence PCR reaction program was as follows: decontamination program 50 ℃ 5 min, 1 cycle; Hoiding Stage program 95 ℃ 5 min, 1 cycle; Cycling Stage 95 ℃ 15 s, 60 ℃ 30 s, 45 cycles. Fluorescence signals were collected at 60 ℃ 30 s, and the reaction results were observed using a fluorescence PCR instrument.
[0046] 1.3 Construction of positive plasmids
[0047] The pol sequence of turtle herpesvirus type I was ligated into the pUC57 vector to construct a positive plasmid for turtle herpesvirus type I.
[0048] 2. Performance testing of the two primer-probe sets
[0049] Using the turtle herpesvirus type I positive plasmid as a DNA template, and referring to Table 1, a real-time fluorescent PCR reaction system was constructed using primer and probe sets TH-M1 and TH-M2, respectively. The real-time fluorescent PCR reaction was performed according to the real-time fluorescent PCR reaction program.
[0050] 3. Interpretation Method
[0051] If the test sample has a Ct value ≥ 45 or no Ct value, and the curve is a straight line or a slightly sloping line, without an "S"-shaped amplification curve, it can be determined that the sample does not contain turtle herpesvirus type I or the content is lower than the detection limit.
[0052] If the Ct value of the test sample is <45 and the curve shows an "S"-shaped amplification curve, it can be determined that the sample contains turtle herpesvirus type I.
[0053] 4. Experimental Results
[0054] The results are as follows Figure 1 As shown, all primers were able to amplify the amplification. The primer-probe set TH-M1 had a smaller Ct value and better reproducibility, and no nonspecific amplification was observed in the negative results. Therefore, the primer-probe set TH-M1 was determined to be the optimal primer-probe set. The following experiments will use the primer-probe set TH-M1 as the primer and probe for real-time fluorescent PCR.
[0055] Example 2: Specificity Experiment
[0056] 1 Experimental Methods
[0057] DNA was extracted from samples positive for Aeromonas hydrophila, Mycoplasma guildrums, Coccidia guciformis, Trypanosoma japonicum, Chinese soft-shelled turtle iridovirus, Chinese soft-shelled turtle hemorrhagic disease virus, Chinese soft-shelled turtle yellow virus, and Elizabethan bacillus meningitidis. The purity and concentration of the DNA samples were determined using a full-wavelength micro-spectrophotometer and stored at -20°C for later use.
[0058] Referring to Table 1, a real-time fluorescence PCR reaction system was constructed using the TH-M1 primer and probe set, and real-time fluorescence PCR reactions were performed on the above positive samples according to the reaction procedure in 1.2.
[0059] 2. Experimental Results
[0060] The results are as follows Figure 2 The results showed that no amplified fluorescent signals were detected in samples positive for Aeromonas hydrophila, Mycoplasma cypriniformis, Coccidia guciformis, Trypanosomiasis, Chinese soft-shelled turtle iridovirus, Chinese soft-shelled turtle hemorrhagic disease virus, Chinese soft-shelled turtle flavivirus, and Elizabethan meningitidis, indicating that the established real-time fluorescent PCR reaction system had good specificity.
[0061] Example 3: Sensitivity and Repeatability Experiments
[0062] 1. Experimental Methods
[0063] The DNA concentration of a plasmid with a plasmid concentration of 100 ng / μL for turtle herpesvirus type I positive plasmid was converted to copy number, resulting in a DNA copy number of 1 × 10⁻⁶. 10 copies / μL, serially diluted 10-fold with TE buffer to obtain 1×10 9 copies / μL, 1×10 8 copies / μL, 1×10 7 copies / μL, 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 copies / μL, 1×10 -1 Plasmids with a concentration of 1×10 copies / μL were selected. 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 copies / μL, 1×10 -1DNA templates were prepared at a concentration of copies / μL, with three replicates for each concentration. The real-time fluorescence PCR reaction system was constructed using the TH-M1 primer and probe set as shown in Table 1. Real-time fluorescence PCR reactions were performed on the above DNA templates according to the reaction procedure in 1.2.
[0064] 2. Experimental Results
[0065] The results are as follows Figure 3 The results showed a typical amplification curve, and the detection sensitivity of this method for positive plasmids reached 1×10⁻⁶. 0 The results of seven dilution gradients and three parallel experiments for each dilution gradient are shown in Table 3. The coefficients of variation range from 0.49% to 1.91%, indicating that the established real-time fluorescence PCR detection method has good reproducibility.
[0066] Table 2. Reproducibility tests of the primer and probe sets for baleen herpesvirus type I.
[0067] Example 4 Negative Stability Test
[0068] 1 Experimental Methods
[0069] Using ddH2O as a negative control, the real-time fluorescence PCR reaction was repeated 20 times, with an additional positive control reference, to perform a negative stability experiment.
[0070] 2 Experimental Results
[0071] The results are as follows Figure 4 The results showed that the established real-time fluorescence PCR reaction system had good negative stability and no non-specific amplification occurred.
[0072] Example 5: Detection of turtle herpesvirus type I in actual samples
[0073] 1 Experimental Methods
[0074] DNA was extracted from 20 actual samples. A real-time fluorescent PCR reaction system was constructed using the primer and probe set TH-M1 as shown in Table 1. Real-time fluorescent PCR was performed on the DNA of each sample to detect whether it was infected with turtle herpesvirus type I.
[0075] 2. Experimental Results
[0076] The results are as follows Figure 5 As shown in the figure, one actual sample tested positive for turtle herpesvirus type I in DNA; 19 actual samples tested negative for turtle herpesvirus type I in DNA.
[0077] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.
Claims
1. A primer set for the specific detection of turtle herpesvirus type I in animals, characterized in that, include: The forward primer TH-M1-F has the sequence shown in SEQ ID NO: 1; The reverse primer TH-M1-R has the sequence shown in SEQ ID NO: 2; The fluorescent probe TH-M1-P has the sequence shown in SEQ ID NO: 3; The fluorescent probe TH-M1-P is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.
2. The primer set for specific detection of turtle herpesvirus type I in animals according to claim 1, characterized in that, The fluorescent probe TH-M1-P is labeled with the fluorescent group FAM at its 5' end and the quencher group MGB at its 3' end.
3. The use of a primer set as described in claim 1 or 2 for the specific detection of turtle herpesvirus type I in animals in the preparation of a kit.
4. The application according to claim 3, characterized in that, The kit also includes PCR reaction premix, positive control reference, negative control reference and / or ddH2O.
5. The application according to claim 4, characterized in that, The positive control reference is a positive plasmid containing the pol sequence of turtle herpesvirus type I, and the negative control reference is ddH2O.
6. A real-time fluorescent PCR reaction system using the primer set for specific detection of turtle herpesvirus type I in animals as described in claim 1 or 2, characterized in that, The total volume is 25 μL, including 12.5 μL of 2× PCR reaction premix, 1 μL of 10 μM forward primer TH-M1-F, 1 μL of 10 μM reverse primer TH-M1-R, 0.5 μL of 10 μM fluorescent probe, 5 μL of DNA template, and 5 μL of ddH2O.
7. The real-time fluorescence PCR reaction system according to claim 6, characterized in that, The 2× reaction premix comprises: Tris-HCl (pH 8.3–8.8) 50 mM, 50mM, 5 mM, dNTPs 200 µM, Taq DNA Polymerase 0.1 U / µL, BSA / glycerol 0.5 mg / mL / 10% v / v, surfactant 0.1%.