Single cross primer pair for rahnella aquatica OmpA gene and application of isothermal amplification rapid detection method
By designing a single-cross primer pair for the OmpA gene of *Laenia aquaticis* and its in vitro isothermal amplification method, the problem of rapid detection of *Laenia aquaticis* was solved, achieving rapid and accurate detection results suitable for field applications.
Patent Information
- Application Number
- CN202510452190.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-11-14
AI Technical Summary
There are few reports on rapid detection methods for aquatic Ranunculosis in the existing technology. The operation steps are cumbersome and the detection cycle is long, which makes it impossible to achieve rapid detection.
A single-cross primer pair for the OmpA gene of *Laenia aquaticis* and its in vitro isothermal amplification method were designed. Specific primer pairs were designed using the target sequence of the OmpA gene, and amplification was performed in a water bath at 62-65℃ using Bst DNA polymerase, dNTPs, betaine, etc. The amplification products were analyzed by electrophoresis.
It enables rapid and accurate detection of aquatic Ranunculosis bacteria, with high sensitivity and simple operation. It does not require an expensive PCR instrument; a water bath is all that is needed to complete the test, making it suitable for on-site testing.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to the application of a single cross primer pair for the OmpA gene of aquatic Ranunculus lanceolata and its rapid in vitro isothermal amplification detection method. Background Technology
[0002] Aquatic Laenella ( Rahnella aquatilis *Laenia* is a Gram-negative facultative anaerobic bacterium belonging to the genus *Laenia* of the family Enterobacteriaceae. It is a newly emerging pathogenic bacterium affecting fish, causing enteritis and septicemia in humans and various aquatic animals, resulting in significant economic losses to the aquaculture industry. Currently, this bacterium has infected a variety of aquatic animals, such as the Chinese forest frog (*L. spp.*). Rana chensinensis ), Damselfish ( Stegastes beebei ) and crucian carp ( Carassius auratus Recent research on rapid detection methods for *Laenia aquaticis* has attracted much attention, as a solution for the clinical diagnosis of this disease is urgently needed. However, there are very few reports on rapid detection and diagnostic techniques for this disease, highlighting the urgent need to develop detection methods for *Laenia aquaticis* suitable for field applications. Summary of the Invention
[0003] Therefore, this invention provides a method for rapid detection of the OmpA gene of Aquatic Ranunculus lanceolata using a single cross primer pair and its in vitro isothermal amplification, in order to solve the problems of cumbersome operation steps, long detection cycle, and inability to achieve rapid detection.
[0004] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: A method for rapid detection of the OmpA gene of *Laenia aquaticis* using a single-cross primer pair and isothermal amplification is disclosed. The method uses the *Laenia aquaticis* OmpA target gene sequence as the DNA detection target and designs specific primer pairs based on its conserved sequence, as shown in SEQ ID NO. 1~5. OmpA gene primer pair 1_5F: 5'-TGGGTGGTATGGTTTGGC-3' (SEQ ID NO.1) OmpA gene primer pair 1_3R: 5'-GCATTGCGTTATCAGGAC-3' (SEQ ID NO.2) OmpA gene cross-primer 2_PF: 5'-CAACGGTGCTAATGGTCGCACCAACTGCTGCCAGTGGAG-3' (SEQ ID NO.3) OmpA gene primer pair 3_P1: 5'-CAACGGTGCTAATGGTCG-3' (SEQ ID NO.4) OmpA gene primer pair 3_P2: 5'-CGTAAGTGATCATGACACTG-3' (SEQ ID NO.5) Preferably, the starting position of the OmpA gene cross primer 2_PF is between 400-500 bp, and the primer sequence of the OmpA gene primer pair 3_P1 is composed of a Tm value between 62-65℃.
[0005] Preferably, the fragment length amplified between primer pairs 1_5F and 3R of the OmpA gene is 200-220 bp.
[0006] Preferably, the intermediate core region amplified by primers 3_P1 and P2 of the OmpA gene primer pair is between 30-50 bp.
[0007] The present invention also provides the application of the above-described specific primer pairs in the identification and / or detection of aquatic Ranuncula for non-disease diagnostic purposes.
[0008] Preferably, the detection method further includes dNTPs, Bst DNA polymerase, 10×Thermopol Buffer, MgSO4, betaine, etc.
[0009] Preferably, the amplification system for the OmpA gene single-cross primer isothermal amplification method is as follows: 20 μL reaction volume: 2 μL 10×Thermopol Buffer; 0.2 μL OmpA gene primer pair 1_5F, 0.2 μL OmpA gene primer pair 1_3R, 1.2 μL OmpA gene cross primer 2_PF, 0.6 μL OmpA gene primer pairs 3_P1 and P2, 1 μL Bst DNA polymerase, 0.6 μL dNTPs, 8 μL betaine, 0.2 μL MgSO4, 3 μL DNA to be detected, and ddH2O added to make up to 20 μL.
[0010] Preferred reaction conditions for the OmpA gene single-cross primer isothermal amplification method are: reaction in a water bath at 62-65℃ for 60 min, followed by inactivation at 80℃ for 5 min.
[0011] The analysis of the amplification products of the present invention is as follows: if the electrophoretic band is a trapezoidal band, it is positive; if the electrophoresis does not show a trapezoidal band, it is negative; the smallest two products are 64bp and 82bp, respectively.
[0012] The present invention has the following advantages: (1) This invention uses the OmpA target gene sequence of Aquatic Larn as the DNA detection target. Based on its conserved sequence, specific primers for the OmpA gene single cross primer isothermal amplification method are designed, which have the advantages of high specificity and high sensitivity; (2) The reaction time is short and amplification can be completed without high temperature denaturation; (3) The whole process does not require an expensive PCR instrument, and only a water bath or constant temperature metal bath is needed to complete the amplification; (4) A detection method for Aquatic Larn has also been established, which can be used to quickly and accurately detect Aquatic Larn in clinical applications. It provides an efficient and rapid technical means for the detection of Aquatic Larn OmpA gene target sequence or DNA target molecular diagnosis, and has important application value for the quarantine, molecular diagnosis and epidemiological monitoring of diseases caused by Aquatic Larn in clinical practice. Attached Figure Description
[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0014] Figure 1 The results show the amplification results of the single-cross primer isothermal amplification method for the OmpA target gene of *R. aquaticus* according to the present invention.
[0015] Figure 2 This is the result of optimizing the Mg2+ concentration in the reaction system of this invention.
[0016] Figure 3 The results show the amplification of DNA from *Laenia aquaticis* and seven other pathogens using the primers of this invention.
[0017] Figure 4 The results of sensitivity analysis for detecting aquatic R. lanuginosa using the OmpA gene single-cross primer isothermal amplification method.
[0018] Figure 5 The results of testing clinical samples using the OmpA gene single-cross primer isothermal amplification method. Detailed Implementation
[0019] Modifications or substitutions to the details and form of the technical solutions of this invention may be made without departing from the spirit and scope of this invention, but all such modifications or substitutions fall within the protection scope of this invention. Unless otherwise specified, the raw materials and chemical reagents used in the embodiments are all commercially available products, and the technical means used are conventional means known to those skilled in the art.
[0020] Example 1: Primer design and synthesis for isothermal amplification of the OmpA gene using single-cross primers for detecting Aquatic Rahn's bacterium. 1.1 Samples and Reagents Bacterial genomic DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; Bst DNA polymerase, Thermopol Buffer, etc. were purchased from NEB.
[0021] 1.2 Primer Design and Synthesis Based on the target sequence of the OmpA gene in *Laenia aquaticis*, single-cross primers for the OmpA gene and specific primer pairs for its isothermal amplification method were designed using Primer Premier 5.0 software. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown below: OmpA gene primer pair 1_5F: 5'-TGGGTGGTATGGTTTGGC-3' (SEQ ID NO.1) OmpA gene primer pair 1_3R: 5'-GCATTGCGTTATCAGGAC-3' (SEQ ID NO.2) OmpA gene cross-primer 2_PF: 5'-CAACGGTGCTAATGGTCGCACCAACTGCTGCCAGTGGAG-3' (SEQ ID NO.3) OmpA gene primer pair 3_P1: 5'-CAACGGTGCTAATGGTCG-3' (SEQ ID NO.4) OmpA gene primer pair 3_P2: 5'-CGTAAGTGATCATGACACTG-3' (SEQ ID NO.5) Example 2: Isothermal amplification of the OmpA gene of *Laenia aquaticis* using specific primers with single-cross primers. Using *Laenia aquaticis* nucleic acid and sterile water as the DNA to be detected, OmpA gene was amplified at isothermally using single-cross primers, with sterile water serving as a negative control. The reaction mixture (20 μL) consisted of: 2 μL of 10×Thermopol Buffer; 0.2 μL of OmpA gene primer pair 1_5F, 0.2 μL of OmpA gene primer pair 1_3R, 1.2 μL of OmpA gene cross primer 2_PF, 0.6 μL of OmpA gene primer pair 3_P1, 0.6 μL of OmpA gene primer pair 3_P2, 1 μL of Bst DNA polymerase, 0.6 μL of dNTPs, 8 μL of betaine, 0.2 μL of MgSO4, and 3 μL of the DNA to be detected, with ddH2O added to bring the total to 20 μL. The reaction conditions were: 62-65℃ water bath for 60 min, followed by inactivation at 80℃ for 5 min. After the PCR reaction is complete, take 5 μL of the PCR product for 2% agarose gel electrophoresis analysis.
[0022] The results showed that only the *Laenia aquaticis* samples amplified ladder-like bands, while the negative control showed no amplification. Figure 1 As shown, M: DNA marker, 1: positive control for Aquatic Laryn bacillus, 2: negative control (sterile water), indicating that the OmpA gene single-cross primer isothermal amplification method can detect nucleic acid samples of Aquatic Laryn bacillus.
[0023] Example 3: Optimization of Mg2+ concentration in the reaction system of the OmpA gene single-cross primer isothermal amplification method The Mg2+ concentration in the reaction system was optimized. Using the reaction system and conditions in Example 2, only the Mg2+ concentration was changed to 2, 3, 4, 5, 6, 7, and 8 mM. Then, the OmpA gene was amplified by single-cross primers at an isothermal temperature and gel electrophoresis was performed. The results were analyzed.
[0024] The results showed that no amplified bands were observed when the Mg2+ concentration was 2 mM, while ladder-like bands amplified when the Mg2+ concentration was 3-8 mM. Furthermore, the band brightness gradually decreased with increasing Mg2+ concentration. Therefore, the optimal Mg2+ concentration was 3 mM. The optimization results of the Mg2+ concentration in the reaction system are shown below. Figure 2 As shown, M: DNA marker, 2-8: Mg2+ concentrations of 2-8 mM, N: negative control (sterile water). The trapezoidal band was brightest and the conditions were best when the Mg2+ concentration was 3 mM.
[0025] Example 4: Optimization of dNTP concentration in the reaction system of the OmpA gene single-cross primer isothermal amplification method The concentration of dNTPs in the reaction system was optimized by using the reaction system and conditions in Example 2, with only the dNTP concentrations changed to 0.3, 0.6, 0.8, 1, 1.2, and 1.6 mM, while keeping other conditions unchanged. Then, the OmpA gene was amplified by single-cross primer isothermal reaction and gel electrophoresis were performed, and the results were analyzed.
[0026] The results showed that the bands were brightest when the dNTP concentration was 0.3 mM. As the concentration increased, the bands became weaker or even disappeared. Therefore, 0.3 mM is the optimal dNTP concentration.
[0027] Example 5: Optimization of betaine concentration in the reaction system of the OmpA gene single-cross primer isothermal amplification method The concentration of betaine in the reaction system was optimized. The reaction system and reaction conditions in Example 2 were used, and only the concentration of betaine was changed to 0.02, 0.12, 0.5, 0.7, 1, and 1.2 M, while other conditions remained unchanged. Then, the OmpA gene was amplified by single cross-primer at a constant temperature and gel electrophoresis was performed, and the results were analyzed.
[0028] The results showed that the bands were brightest and best when the betaine concentration was 1M, so 1M is the optimal betaine concentration.
[0029] Example 6: Optimization of reaction time and reaction temperature for isothermal amplification of the OmpA gene using a single cross-primer Using the reaction system and conditions described in Example 2, only the reaction temperature was changed to 57, 59, 61, 63, 65, and 67°C, while other conditions remained unchanged; similarly, only the reaction time was changed to 30, 45, 60, 75, and 90 min, while other conditions remained unchanged. Then, the OmpA gene was subjected to isothermal amplification reaction using single-cross primers and gel electrophoresis, and the results were analyzed.
[0030] The results showed that the optimal reaction temperature was 62-65℃ and the optimal reaction time was 60 min.
[0031] Example 7: Specificity detection of the OmpA gene isothermal amplification method using single-cross primers. Aquatic Laryn bacilli were extracted using a bacterial genomic DNA extraction kit. Rahnella aquatilis Aeromonas hydrophila ( ) Aeromonas hydrophila Aeromonas versicolor ( Aeromonas veronii Aeromonas heterophile ( ) Aeromonas allosaccharophila Providencia retaliculata ( ), Providencia rettgeri ), Alphakine ( Vibrio alfacsensis Yersinia ruvidii ( Yersinia ruckeri Vibrio vulnificus ( Vibrio vulnificus The DNA of OmpA was amplified at isothermal temperature using a single-cross primer amplification reaction with the reaction system and conditions described in Example 2. The results are as follows: Figure 3 As shown, M: DNA marker, 1: Aquatic Laryn bacillus, 2-8: Other pathogens, 9: Negative control. Only Aquatic Laryn bacillus could amplify a ladder-shaped band, while other pathogens and the negative control did not amplify, indicating that the OmpA gene single-cross primer isothermal amplification method has good specificity.
[0032] Example 8: Sensitivity detection of *Laenia aquaticis* using the OmpA gene single-cross primer isothermal amplification method. The DNA of *Laenia aquaticis* was serially diluted 10-fold to approximately 48 μg, 4.8 μg, 480 ng, 48 ng, 4.8 ng, 480 pg, 48 pg, and 4.8 pg / ml. These diluted DNAs were then amplified using the OmpA gene single-crossover primer isothermal amplification method. The results are as follows: Figure 4As shown, M: DNA marker; 1-8: DNA concentrations of *Laenia aquaticis* (48 μg, 4.8 μg, 480 ng, 48 ng, 4.8 ng, 480 pg, 48 pg, 4.8 pg / ml, respectively); 9: negative control. The results indicate that the OmpA gene single-cross primer isothermal amplification method of this invention has a minimum detection limit of 48 pg / ml for *Laenia aquaticis*, demonstrating high sensitivity.
[0033] Example 9: Detection of clinical samples using the OmpA gene single-cross primer isothermal amplification method. The OmpA gene isothermal amplification method using single-cross primers of this invention was used to detect the visceral tissues of crucian carp infected with *Laenia aquaticis*. Nucleic acid DNA was extracted from the liver, spleen, anterior kidney, somatic kidney, foregut, midgut, hindgut, and gills of the crucian carp using a bacterial genomic DNA extraction kit. Then, the OmpA gene wasothermal amplification reaction using single-cross primers was performed using the reaction system and conditions described in Example 2. After the PCR reaction was completed, 5 μL of the PCR product was analyzed by 2% agarose gel electrophoresis. The results are as follows: Figure 5 As shown, M: DNA marker; 1-8: liver, spleen, anterior kidney, somatic kidney, foregut, midgut, hindgut, and gills; 9: negative control. The results indicate that all clinical samples were positive, and the negative control was negative, demonstrating that the OmpA gene single-cross primer isothermal amplification method of this invention can be used for the detection of clinical samples.
[0034] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A specific primer sequence pair for detecting *Laenia aquaticis*, characterized in that, Using the OmpA gene sequence of *Laenia aquaticis* as the DNA detection target, five specific primer pairs were designed based on its conserved sequence, as shown in SEQ ID NO. 1~5: OmpA gene primer pair 1_5F: 5'-TGGGTGGTATGGTTTGGC-3' (SEQ ID NO.1) OmpA gene primer pair 1_3R: 5'-GCATTGCGTTATCAGGAC-3' (SEQ ID NO.2) OmpA gene cross-primer 2_PF: 5'-CAACGGTGCTAATGGTCGCACCAACTGCTGCCAGTGGAG-3' (SEQ ID NO.3) OmpA gene primer pair 3_P1: 5'-CAACGGTGCTAATGGTCG-3' (SEQ ID NO.4) OmpA gene primer pair 3_P2: 5'-CGTAAGTGATCATGACACTG-3' (SEQ ID NO.5).
2. The specific primer pair as described in claim 1, characterized in that, The starting position of the OmpA gene cross primer 2_PF is between 400-500 bp, and together with the primer sequence of the OmpA gene primer pair 3_P1, the Tm value is between 62-65℃.
3. The specific primer pair as described in claim 1, characterized in that, The fragment length amplified between primer pairs 1_5F and 3R for the OmpA gene is 200-220 bp.
4. The specific primer pair as described in claim 1, characterized in that, The amplified core region of the OmpA gene using primers 3_P1 and P2 ranged from 30 to 50 bp.
5. The application of the specific primer pair as described in claim 1 in the identification and / or detection of aquatic Ranuncula for non-disease diagnostic purposes.
6. The application as described in claim 5, characterized in that, The detection method also includes dNTPs, Bst DNA polymerase, 10×Thermopol Buffer, MgSO4, betaine, etc.
7. The application as described in claim 5, characterized in that, The reaction system for the detection reagent or the OmpA gene single cross primer isothermal amplification method is as follows: 20 μL reaction system: 2 μL 10×Thermopol Buffer; 0.2 μL OmpA gene primer pair 1_5F, 0.2 μL OmpA gene primer pair 1_3R, 1.2 μL OmpA gene cross primer 2_PF, 0.6 μL OmpA gene primer pairs 3_P1 and P2, 1 μL Bst DNA polymerase, 0.6 μL dNTPs, 8 μL betaine, 0.2 μL MgSO4, 3 μL DNA to be detected, and ddH2O added to make up to 20 μL.
8. The application as described in claim 5, characterized in that, The reaction conditions for the reagent or OmpA gene single cross primer isothermal amplification method are: react in a water bath at 62-65℃ for 60 min, and then inactivate at 80℃ for 5 min.
9. The application as described in claim 5, characterized in that, The analysis of the amplification products was as follows: if the electrophoretic band is a trapezoidal band, it is positive; if the electrophoresis band is not a trapezoidal band, it is negative; the smallest two products were 64 bp and 82 bp, respectively.