Vibrio mediterraneus tcpB gene fluorescent quantitative PCR (polymerase chain reaction) identification primer and method
By designing specific primers and combining fluorescence quantitative PCR technology, the samples of tcpB gene detection of Vitiligo Mediterranean tcpB gene in the prior art were solved, and the samples of tcpB gene detection were strictly required, cumbersome operation procedures, poor sensitivity and reduced specificity were achieved, and the rapid, accurate and specificity detection of the tcpB gene was achieved, with high sensitivity and high specificity.
Patent Information
- Application Number
- CN202510073276.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2025-04-11
AI Technical Summary
In the prior art, when detecting whether Vitiligo Mediterranean carries the tcpB gene, there are problems such as strict sample purity and concentration requirements, cumbersome operation procedures, poor sensitivity and reduced specificity.
The fluorescence quantitative PCR identification primers of the V. Mediterranean tcpB gene were designed. Through fluorescence quantitative PCR technology, specific primers tcpB-F and tcpB-R were used for detection, and combined with cloning vectors and positive cloning detection primers, the rapid and accurate detection of the tcpB gene was achieved.
The rapid, accurate and specific detection of the V. Mediterranean tcpB gene is achieved, with high sensitivity and specificity, simplifying the operation process and improving the intuitiveness of the detection results.
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Figure CN119799931A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to a primer and method for identifying the tcpB gene of Vibrio mediterranei by fluorescence quantitative PCR (Real-time fluorescent quantitative PCR, qPCR). Background Art
[0002] The tcpB gene of Mediterranean Vibrio belongs to the toxin coregμLated pilus (TCP) gene cluster. In Vibrio cholerae, the tcpB gene encodes the structural protein of type IV pili, forming the tiny pili at the top of the toxin coregμLated pilus, and at the same time assists the bacteria to capture CTXΦ and convert it into CTX. Tcp is one of the important virulence factors of Vibrio cholerae and Vibrio mimicus, assisting the functional expression of CTX secretion, causing severe diarrhea in the host; Tcp is also one of the adhesins, with tissue cell adhesion and hemagglutination; Tcp is also an important protective antigen. At the same time, the pathogenicity of Tcp of pathogenic Vibrio has been confirmed by animal experiments.
[0003] Mediterranean Vibrio was reported as a pathogen of Vibrio in shellfish in 2023. Brown or black spots appeared on the surface of the shells of dying juvenile razor clams, and they had other typical clinical symptoms of vibriosis, such as weak contraction of the adductor muscles of juvenile shellfish and incomplete shell closure. Stress experiments found that it is also pathogenic to oyster larvae and mud clam larvae, and is considered to be a disease that is more harmful to shellfish seedlings. In addition, a large number of clinical biological tests have found that Mediterranean Vibrio carrying the TCP-PAI pathogenicity island has a higher pathogenicity to shellfish. Therefore, TCP-PAI has also become an important target for detecting the virulence of Mediterranean Vibrio, and one of its structural proteins, tcpB, has become an important target gene. In order to achieve the goal of quickly determining whether Mediterranean Vibrio carries the tcpB gene, traditional polymerase chain reaction (PCR) technology has been applied to the monitoring process of Mediterranean Vibrio TCP gene carrying. However, conventional PCR technology has several limitations: first, it has strict requirements on the purity and concentration of samples and is extremely susceptible to contamination. Even trace amounts of exogenous contamination may lead to false positive results. Second, the implementation of the experiment requires a formal laboratory environment, and specimen collection, nucleic acid extraction, amplification, and product analysis must be carried out in separate rooms, making the operation process cumbersome and complex. Third, conventional PCR technology has poor sensitivity, making it difficult to perform accurate quantitative analysis of the target object. Fourth, conventional PCR is difficult to effectively distinguish between PCR products of similar size, which reduces the specificity of the detection method. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a fluorescent quantitative PCR identification primer and method for the tcpB gene of Mediterranean Vibrio in view of the deficiencies of the prior art. The method of the present invention is simple to operate, the result is intuitive, and it has the significant advantages of high sensitivity and high specificity. It can realize rapid and accurate specific detection of the tcpB gene of Mediterranean Vibrio, detect and judge whether Mediterranean Vibrio carries the tcpB gene at the DNA level, and is of great significance for investigating the prevalence of Mediterranean Vibrio tcpB and establishing effective disease prevention and control measures in a targeted manner.
[0005] The technical solution adopted by the present invention to solve the above technical problems is: fluorescent quantitative PCR identification primers for the tcpB gene of Vibrio mediterranei, the sequences of the primers are as follows:
[0006] tcpB-F: 5'-AGGGGTTCCGCTTTACTTGACA-3';
[0007] tcpB-R: 5'-ACCTATCGTTTGTCCAGCCG-3'.
[0008] The method for identifying the tcpB gene of Mediterranean Vibrio by fluorescence quantitative PCR uses tcpB-F and tcpB-R as specific primers, and performs qualitative detection of the tcpB gene of Mediterranean Vibrio by fluorescence PCR detection according to the Ct value and amplification curve, wherein the sequences of the primers are as follows:
[0009] tcpB-F: 5'-AGGGGTTCCGCTTTACTTGACA-3';
[0010] tcpB-R: 5'-ACCTATCGTTTGTCCAGCCG-3'.
[0011] Furthermore, the above-mentioned Vibrio mediterranei tcpB gene fluorescence quantitative PCR identification method specifically comprises the following steps:
[0012] (1) Sample collection and processing
[0013] The genomic DNA of the Vibrio mediterranei samples was extracted using a DNA extraction kit;
[0014] (2) PCR detection
[0015] The extracted genomic DNA was amplified by PCR using the designed specific primers tcpB-F and tcpB-R. After the amplification reaction, 7 μL of the PCR amplification product was taken and detected by 1% agarose gel electrophoresis. The electrophoresis detection results were then tested to determine whether the PCR amplification product was consistent with the target fragment. If consistent, the PCR amplification product was purified and used as the target gene tcpB.
[0016] (3) Select the PBR322 cloning vector, select the double restriction sites BamHI and NruI according to the tcpB gene, and digest the PBR322 cloning vector; design homology arm primers according to the tcpB gene and the double restriction sites BamHI and NruI, and connect the target gene tcpB with the PBR322 cloning vector using the seamless cloning kit ABclonal;
[0017] (4) Design positive clone detection primers 322-F: 5′-TCACTATGGCGTGCTGC-3′ and 322-R: 5′-GCTGTCCCTGATGGTCGT-3′, and screen positive clone strains by ordinary PCR;
[0018] (5) Detecting positive clones by PCR and sequencing to detect whether the target gene tcpB is connected to the vector and transformed successfully, amplifying and culturing the positive clones that have been transformed successfully, extracting the plasmids of the positive strains, performing fluorescence quantitative PCR after gradient dilution, and preparing a standard curve;
[0019] (6) Streak and revive the Mediterranean Vibrio strains, pick up single colonies on the recovery plate, add them to liquid culture medium for amplification and culture for 14 hours, and then use ordinary PCR to detect whether the strains carry the Mediterranean Vibrio tcpB gene. For strains carrying the Mediterranean Vibrio tcpB gene, extract their DNA, and perform fluorescent quantitative PCR detection after passing the quality inspection. The Mediterranean Vibrio tcpB gene is qualitatively detected based on the Ct value of the standard curve and the amplification curve. If the Ct value of the strain is less than 32 and an exponential amplification curve appears, it is determined that the strain contains the Mediterranean Vibrio tcpB gene.
[0020] Further, the reaction system of the fluorescent quantitative PCR described in step (5) is composed of: 2×UniversalSYBR Green Fast qPCR Mix: 10 μL, upstream primer F: 0.5 μL, downstream primer R: 0.5 μL, DNA template: 1 μL, sterile water: 8 μL; the reaction procedure of the fluorescent quantitative PCR described in step (5) includes: pre-denaturation: 95°C, 3 min; denaturation: 95°C, 10 s, renaturation: 58.5°C, 30 s, 40 cycles; melting curve: 65°C, 5 s, 95°C, 50 s. 2×Universal SYBRGreen Fast qPCR Mix is a dye with simple operation, high sensitivity, wide application range and economical benefits. When the dye is combined with dsDNA, the fluorescent signal will be enhanced by 800 to 1000 times, and its sensitivity is 25 to 100 times higher than that of EB dye, and its toxicity is lower than that of EB dye. Compared with fluorescent probes, the use of 2×Universal SYBR Green Fast qPCR Mix dye does not require the design of specific probes and their matching primers, which can significantly shorten experimental time and scientific research expenses.
[0021] Furthermore, the concentrations of the gradient dilutions in step (5) are: 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 per / μL.
[0022] Compared with the prior art, the present invention has the following advantages: the present invention designs specific primers tcpB-F and tcpB-R, utilizes common PCR technology to amplify the tcpB coding gene in segments, selects double restriction sites according to the cloning vector after amplification is correct, designs homology arm primers, designs positive clone detection primers to screen positive clone strains, extracts positive strain plasmids after sequencing is correct, performs fluorescence quantitative PCR after gradient dilution, prepares a standard curve, and then performs fluorescence quantitative PCR detection on the DNA of the strain carrying the Mediterranean Vibrio tcpB gene, thereby detecting and judging whether the Mediterranean Vibrio carries the tcpB gene at the DNA level. The method of the present invention is simple to operate, the result is intuitive, and has the significant advantages of high sensitivity and high specificity, and can realize rapid and accurate specific detection of the Mediterranean Vibrio tcpB gene, which is of great significance for investigating the prevalence of Mediterranean Vibrio tcpB and establishing effective disease prevention and control measures in a targeted manner. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1This is the result of common PCR test of Mediterranean Vibrio strain samples;
[0024] Figure 2 The standard curve of the copy number of different concentration gradient standards detected by fluorescent quantitative PCR in the embodiment;
[0025] Figure 3 The amplification curve of the copy number of the standard product with different concentration gradients detected by fluorescent quantitative PCR in the embodiment;
[0026] Figure 4 It is the amplification curve of the samples of DT01, DT02, DT03, DT04, DT05, DT06 and NTC group in the embodiment. DETAILED DESCRIPTION
[0027] The present invention is further described in detail below with reference to the accompanying drawings.
[0028] Example:
[0029] According to the conserved region of the tcpB gene and the requirements for primer design of fluorescent quantitative PCR, the primers for fluorescent quantitative PCR were designed using Primer5.0 software: tcpB-F: 5'-AGGGGTTCGCTTTACTTGACA-3', tcpB-R: 5'-ACCTATCGTTTGTCCAGCCG-3', amplified fragment: 128 bp, synthesized by Shanghai Sangon Biotechnology Co., Ltd.
[0030] The Mediterranean Vibrio strains of our laboratory were streaked to recover, and single colonies on the recovery plates were picked up and added to 2216E liquid culture medium for amplification and culture for 14 hours. Conventional PCR was used to detect whether the strains carried the Mediterranean Vibrio tcpB gene. According to the test results, 7 Mediterranean Vibrio strain samples were prepared and named DT01, DT02, DT03, DT04, DT05, DT06, and DT07, respectively. Among them, DT01, DT02, and DT03 did not carry the tcpB gene, while DT04, DT05, DT06, and DT07 carried the tcpB gene. Figure 1 This is the result of common PCR detection of Mediterranean Vibrio strain samples, Lane M: 500bp DNA ladder marker.
[0031] Using the designed primers tcpB-F and tcpB-R as specific primers, the tcpB gene of Vibrio mediterranei was qualitatively detected according to the Ct value and amplification curve by fluorescence PCR detection, which specifically includes the following steps:
[0032] (1) Sample collection and processing
[0033] The genomic DNA of Mediterranean Vibrio samples DT02 and DT07 was extracted using the marine tissue genomic DNA extraction kit of TIANGEN Biotechnology Co., Ltd.;
[0034] (2) PCR detection
[0035] The extracted genomic DNA was amplified by PCR using the designed specific primers tcpB-F and tcpB-R. The PCR amplification was performed using a T20 double-slot gradient PCR instrument. After the amplification reaction was completed, 7 μL of the PCR amplification product was taken and detected by 1% agarose gel electrophoresis. The electrophoresis detection results were then tested by a fully automatic ultraviolet gel imaging analysis system to determine whether the PCR amplification product was consistent with the target fragment. If consistent, the PCR amplification product of the DT07 strain sample was recovered, purified and its concentration was determined, and it was used as the target gene tcpB.
[0036] (3) Select the PBR322 cloning vector, select the double restriction sites BamHI and NruI according to the tcpB gene, and digest the PBR322 cloning vector; design homology arm primers according to the tcpB gene and the double restriction sites BamHI and NruI, and connect the target gene tcpB with the PBR322 cloning vector using the seamless cloning kit ABclonal;
[0037] (4) Design positive clone detection primers 322-F: 5′-TCACTATGGCGTGCTGC-3′ and 322-R: 5′-GCTGTCCCTGATGGTCGT-3′, and screen positive clone strains by ordinary PCR;
[0038] (5) The positive clone strains obtained by screening were sent to Shanghai Sangon Biotechnology Co., Ltd. for PCR detection, and their base sequences were determined. The amplified region of the tcpB gene was compared to detect whether the target gene tcpB was connected to the vector and transformed successfully. The positive clone strains with successful transformation were amplified and cultured. The plasmids of the positive strains were extracted using the Omega plasmid extraction kit and graded diluted 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 per / μL, and then perform fluorescence quantitative PCR to prepare a standard curve. The standard curve of the copy number of different concentration gradient standards detected by fluorescence quantitative PCR is shown in Figure 2 The amplification curves of the copy numbers of the standard products with different concentration gradients are shown in Figure 3 ;
[0039] The reaction volume of the above-mentioned fluorescence quantitative PCR reaction system is 20 μL, and its composition is as follows: 2×Universal SYBRGreen Fast qPCR Mix: 10 μL, upstream primer F: 0.5 μL, downstream primer R: 0.5 μL, DNA template: 1 μL, sterile water: 8 μL; the reaction procedure of fluorescence quantitative PCR includes: pre-denaturation: 95°C, 3 min; denaturation: 95°C, 10 s, renaturation: 58.5°C, 30 s, 40 cycles; melting curve: 65°C, 5 s, 95°C, 50 s;
[0040] (6) DNA was extracted from strains DT04, DT05, and DT06 carrying the tcpB gene of Vibrio mediterranei and strains DT01, DT02, and DT03 not carrying the tcpB gene of Vibrio mediterranei, and fluorescent quantitative PCR was performed after passing the quality inspection. The amplification curves of samples in the DT01, DT02, DT03, DT04, DT05, DT06, and NTC groups are shown in Figure 2. Figure 4 .Depend on Figure 4 It can be seen that the Ct value of the control group NTC is ≥32, and the strains DT01, DT02, and DT03 that do not carry the Mediterranean Vibrio tcpB gene have an amplification curve that is synchronized with the template-free control group NTC. The cycle number of their tcpB gene is around 32, and the fluorescence signal still does not reach the threshold after the fluorescence quantitative PCR amplification cycle exceeds 32 times, that is, the Ct value is ≥32, so the test result is determined to be negative, that is, the strains DT01, DT02, and DT03 do not contain the Mediterranean Vibrio tcpB gene; while the strains DT04, DT05, and DT06 that carry the Mediterranean Vibrio tcpB gene have fluorescence signals that reach the threshold at around 15, 13, and 16 of the fluorescence quantitative PCR amplification cycle, that is, the Ct value is <32, and exponential amplification curves with different cycle numbers appear, so the test result is determined to be positive, that is, the strains DT04, DT05, and DT06 contain the Mediterranean Vibrio tcpB gene. According to the standard curve prepared in step (5), the relevant calculation results for the strains DT04, DT05, and DT06 are shown in Table 1.
[0041] Table 1 Calculation results of tcpB gene copy number
[0042]
Claims
1. A fluorescent quantitative PCR primer for identifying tcpB gene of Vibrio mediterranei, characterized in that: The sequences of the primers are as follows: tcpB-F: 5'-AGGGGTTCCGCTTTACTTGACA-3'; tcpB-R: 5'-ACCTATCGTTTGTCCAGCCG-3'.
2. A fluorescence quantitative PCR identification method for the tcpB gene of Vibrio mediterranei, characterized in that: Using tcpB-F and tcpB-R as specific primers, the tcpB gene of Vibrio mediterranei was qualitatively detected according to the Ct value and amplification curve by fluorescence PCR detection, wherein the sequences of the primers are as follows: tcpB-F: 5'-AGGGGTTCCGCTTTACTTGACA-3'; tcpB-R: 5'-ACCTATCGTTTGTCCAGCCG-3'.
3. The method for identifying the tcpB gene of Vibrio mediterranei by fluorescence quantitative PCR according to claim 2, characterized in that: The specific steps include: (1) Sample collection and processing The genomic DNA of the Vibrio mediterranei samples was extracted using a DNA extraction kit; (2) PCR detection The extracted genomic DNA was amplified by PCR using the designed specific primers tcpB-F and tcpB-R. After the amplification reaction, 7 μL of the PCR amplification product was taken and detected by 1% agarose gel electrophoresis. The electrophoresis detection results were then tested to determine whether the PCR amplification product was consistent with the target fragment. If consistent, the PCR amplification product was purified and used as the target gene tcpB. (3) Select the PBR322 cloning vector, select the double restriction sites BamHI and NruI according to the tcpB gene, and digest the PBR322 cloning vector; design homology arm primers according to the tcpB gene and the double restriction sites BamHI and NruI, and connect the target gene tcpB with the PBR322 cloning vector using the seamless cloning kit ABclonal; (4) Design positive clone detection primers 322-F: 5′-TCACTATGGCGTGCTGC-3′ and 322-R: 5′-GCTGTCCCTGATGGTCGT-3′, and screen positive clone strains by ordinary PCR; (5) Detecting positive clones by PCR and sequencing to detect whether the target gene tcpB is connected to the vector and transformed successfully, amplifying and culturing the positive clones that have been transformed successfully, extracting the plasmids of the positive strains, performing fluorescence quantitative PCR after gradient dilution, and preparing a standard curve; (6) Streak and revive the Mediterranean Vibrio strains, pick up single colonies on the recovery plate, add them to liquid culture medium for amplification and culture for 14 hours, and then use ordinary PCR to detect whether the strains carry the Mediterranean Vibrio tcpB gene. For strains carrying the Mediterranean Vibrio tcpB gene, extract their DNA, and perform fluorescent quantitative PCR detection after passing the quality inspection. The Mediterranean Vibrio tcpB gene is qualitatively detected based on the Ct value of the standard curve and the amplification curve. If the Ct value of the strain is less than 32 and an exponential amplification curve appears, it is determined that the strain contains the Mediterranean Vibrio tcpB gene.
4. The method for identifying the tcpB gene of Vibrio mediterranei by fluorescence quantitative PCR according to claim 3, characterized in that: The reaction system of the fluorescent quantitative PCR described in step (5) is composed of: 2×Universal SYBR Green Fast qPCRMix: 10 μL, upstream primer F: 0.5 μL, downstream primer R: 0.5 μL, DNA template: 1 μL, sterile water: 8 μL; the reaction procedure of the fluorescent quantitative PCR described in step (5) includes: pre-denaturation: 95°C, 3 min; denaturation: 95°C, 10 s, renaturation: 58.5°C, 30 s, 40 cycles; melting curve: 65°C, 5 s, 95°C, 50 s.
5. The method for identifying the tcpB gene of Vibrio mediterranei by fluorescence quantitative PCR according to claim 3, characterized in that: The concentrations of the gradient dilutions in step (5) are: 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 per / μL.