CPA-LFD method for detecting Rahnella aquatica and detection kit
By using the CPA-LFD method and specific primer sequences, the complexity and time-consuming nature of detecting aquatic Ranuncula have been resolved, enabling rapid and accurate detection suitable for on-site testing of human, animal, and fish samples.
Patent Information
- Application Number
- CN202510450626.0
- 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
Existing methods for detecting aquatic Ranunculosis are cumbersome, have long testing cycles, require specialized equipment and techniques, and cannot achieve rapid and simple detection.
The single-cross primer isothermal amplification combined with colloidal gold transverse flow test strip (CPA-LFD) method was adopted. Biotin and 6-carboxyfluorescein were labeled with specific primer sequences P1 and P2 and then combined with nucleic acid detection test strips for detection.
It enables rapid and accurate detection of aquatic Ranger bacteria with a sensitivity of up to 48 fg/μL. It is simple to operate, does not rely on professional equipment, and is suitable for field applications.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a CPA-LFD method and detection kit for rapid detection of aquatic Ranuncula. Background Technology
[0002] Aquatic Laenella ( Rahnella aquatilis *Laenia aquaticis* is a newly emerging infectious disease pathogen that can cause disease in various fish, animals, and humans, leading to enteritis and septicemia. It is also a potential zoonotic pathogen, seriously impacting human health and aquatic animal health, posing a significant threat to the aquaculture industry. Currently, the detection of *Laenia aquaticis* mainly relies on bacterial culture, physiological and biochemical identification, and 16S rDNA molecular identification. These methods are cumbersome, time-consuming, and require high-level experimental conditions, expensive equipment, and skilled personnel, failing to provide simple and rapid detection. Therefore, establishing a simple, rapid, and highly sensitive detection method and developing a *Laenia aquaticis* detection kit suitable for field applications is crucial. Summary of the Invention
[0003] Therefore, this invention provides a CPA-LFD method and test kit for detecting aquatic Ranuncula, namely, a single-cross primer isothermal amplification combined with colloidal gold transverse flow test strip, 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 CPA primer sequence for detecting aquatic Ranunculosis bacteria, comprising two CPA probe primers, P1 and P2, with sequences shown in SEQ ID NO. 1~2: P1: 5'-Biotin-CAACGGTGCTAATGGTCG-3' (SEQ ID NO.1) P2: 5'-FAM-CGTAAGTGATCATGACACTG-3' (SEQ ID NO.2) Preferably, the 5' end of primer P1 is labeled with biotin, and the 5' end of primer P2 is labeled with 6-carboxyfluorescein (6-FAM). Therefore, the amplification product of CPA contains both FAM and biotin labels.
[0005] The present invention also provides the application of the above-mentioned specific primer sequences in the preparation of reagents or kits for the detection of aquatic Ranuncula by the CPA-LFD method.
[0006] Preferably, the kit also includes dNTPs, Bst DNA polymerase, Thermopol Buffer, MgSO4, betaine, etc.
[0007] Preferably, the kit further includes a nucleic acid detection test strip, which is a colloidal gold transverse flow test strip (LFD).
[0008] Preferably, the CPA amplification system is as follows: 20 μL reaction volume: 2 μL 10×Thermopol Buffer; 0.2 μL each of forward and reverse primers 5F and 3R; 1.2 μL single cross primer PF; 0.6 μL CPA probe primer P1; 0.6 μL CPA probe primer P2; 1 μL Bst DNA polymerase; 0.6 μL dNTPs; 8 μL betaine; 0.2 μL MgSO4; 3 μL DNA; and ddH2O added to make up to 20 μL.
[0009] Preferably, the CPA reaction conditions are: reaction in a water bath at 63°C for 45-60 min, followed by inactivation at 80°C for 5 min.
[0010] In this invention, the preferred method of using the kit includes: performing amplification using the above-described amplification system and conditions to obtain amplification products; when not using a nucleic acid test strip, taking 5 μL of the amplification product, performing electrophoresis on a 2% (w / v) agarose gel, and then imaging it in a gel imaging system; if the electrophoresis result shows a characteristic trapezoidal band in CPA, the result is positive; if there are no bands, the result is negative; when using a nucleic acid test strip, taking 10 μL of the amplification product, and testing it with the nucleic acid test strip, if only one red line appears in the control area C, the sample test result is negative; if two red lines appear, one test line and one control line, the sample test result is positive; if no red lines appear in either the control area C or the test area T, the nucleic acid test strip is invalid and the test result is invalid.
[0011] The present invention has the following advantages: (1) It has good specificity and can quickly and effectively detect aquatic Laryn bacilli; (2) Accurate and rapid, it can detect aquatic Ranger bacteria down to 48 fg / μL, and the detection time can be shortened to less than one hour; (3) It is easy to operate, does not rely on professional testing personnel, and has low requirements for instruments. Only a metal bath or constant temperature water bath is needed. (4) The test results are easy to interpret and are simple and intuitive. (The nucleic acid test strip has a control line and a test line. If a red line appears on both the control line and the test line, the result is positive.) (5) It is suitable for rapid detection of aquatic Ranunculosis in human clinical samples, animals, fish and food contamination, and is of great significance for monitoring, diagnosis and epidemiological control of diseases caused by aquatic Ranunculosis. Attached Figure Description
[0012] 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.
[0013] Figure 1 The results of CPA-LFD amplification of the aquatic Ranger bacillus of the present invention are shown.
[0014] Figure 2 This is the result of optimizing the Mg2+ concentration in the reaction system of this invention.
[0015] Figure 3 The results of DNA amplification of *Laenia aquaticis* and seven other pathogens using the CPA-LFD method of this invention are shown.
[0016] Figure 4 The results show the sensitivity analysis of *R. aquaticus* using the CPA-LFD method.
[0017] Figure 5 The results of the CPA-LFD method for detecting Aquatic Ranunculus latum on clinical samples are presented. Detailed Implementation
[0018] 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.
[0019] Example 1: Design and Synthesis of CPA Primers for Detecting Aquatic Laryn Bacteria 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 Company; nucleic acid test strips were purchased from Hangzhou Youstar Company.
[0020] 1.2 Primer Design and Synthesis Based on the conserved sequence of *Laenia aquaticis*, CPA-specific primers were designed using Primer Premier 5.0 software. Primers containing both biotin and FAM-labeled probes, capable of recognizing specific regions of the bacterium's DNA, and their sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown below: P1: 5'-Biotin-CAACGGTGCTAATGGTCG-3' (SEQ ID NO.1) P2: 5'-FAM-CGTAAGTGATCATGACACTG-3' (SEQ ID NO.2) Example 2: Amplification of Aquatic Rahn's bacillus using CPA-LFD with specific primer sequences Using *Laenia aquaticis* nucleic acid and sterile water as templates, CPA amplification was performed separately, with sterile water serving as a negative control. The reaction system (20 μL) consisted of: 2 μL of 10×Thermopol Buffer; 0.2 μL each of forward and reverse primers 5F and 3R; 1.2 μL of single-cross primer PF; 0.6 μL of CPA probe primer P1; 0.6 μL of CPA probe primer P2; 1 μL of Bst DNA polymerase; 0.6 μL of dNTPs; 8 μL of betaine; 0.2 μL of MgSO4; 3 μL of DNA; and ddH2O added to bring the total to 20 μL. The reaction conditions were: 63℃ water bath for 45-60 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; use nucleic acid detection strips for detection, take 5~10 μL of the amplification product and add it to the bottom of the test strip, then add 100 μL of buffer (Hangzhou Youstar Biotechnology Co., Ltd.), and the results can be observed within 5~10 minutes.
[0021] The results showed that only the *Laenia aquaticis* sample amplified a ladder-like band, while the negative control showed no amplification; the nucleic acid test strips showed that the *Laenia aquaticis* sample was positive, while the negative control was negative. Figure 1 As shown, M: DNA marker, 1: positive control for Aquatic Laryn bacillus, 2: negative control (sterile water), indicating that the CPA-LFD method can detect nucleic acid samples of Aquatic Laryn bacillus.
[0022] Example 3 Optimization of Mg2+ concentration in the CPA reaction system of *Laenia aquaticis* The concentration of Mg2+ in the reaction system was optimized. Using the reaction system and conditions in Example 2, only the concentration of Mg2+ was changed to 2, 3, 4, 5, 6, 7, and 8 mM. Then, CPA reaction and gel electrophoresis were performed, and the results were analyzed.
[0023] 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.
[0024] Example 4: Optimization of dNTP concentration in CPA reaction system The concentration of dNTPs in the reaction system was optimized by using the reaction system and reaction conditions in Example 2, changing only the concentration of dNTPs to 0.3, 0.6, 0.8, 1, 1.2, and 1.6 mM, while keeping other conditions unchanged. Then, CPA reaction and gel electrophoresis were performed, and the results were analyzed.
[0025] 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.
[0026] Example 5: Optimization of betaine concentration in the CPA reaction system The concentration of betaine in the reaction system was optimized by using the reaction system and reaction conditions in Example 2, only changing the concentration of betaine to 0.02, 0.12, 0.5, 0.7, 1, and 1.2 M, while keeping other conditions unchanged. Then, CPA reaction and gel electrophoresis were performed, and the results were analyzed.
[0027] The results showed that the bands were brightest and best when the betaine concentration was 1M, so 1M is the optimal betaine concentration.
[0028] Example 6: Optimization of reaction time and temperature in the CPA kit Using the reaction system and conditions 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, CPA reaction and gel electrophoresis were performed, and the results were analyzed.
[0029] The results showed that the optimal reaction temperature was 63℃ and the optimal reaction time was 60 min.
[0030] Example 7 Specificity detection of the CPA-LFD method Genomic DNA was extracted from *Laenia aquaticis*, *Providendum repens*, *Aeromonas hydrophila*, *Alfaxone*, *Aeromonas vesiculosus*, *Yersinia rumeni*, *Vibrio vulnificus*, and *Aeromonas heterotrophus* using a bacterial genomic DNA extraction kit. The DNA was then analyzed using the optimized CPA-LFD method for *Laenia aquaticis* as described in Examples 2 and 3, with a blank control included. Results are as follows: Figure 3 As shown, 1: Aquatic Laryn bacillus, 2-8: Other pathogens, 9: Negative control. The nucleic acid test strip shows that only Aquatic Laryn bacillus is positive, while other pathogens and the negative control are negative, indicating that CPA-LFD has good specificity.
[0031] Example 8: Sensitivity detection of Aquatic Raenella using the CPA-LFD method The DNA of *Laenia aquaticis* was serially diluted 10-fold to approximately 48 ng, 4.8 ng, 480 pg, 48 pg, 4.8 pg, 480 fg, 48 fg, and 4.8 fg / ul. It was then detected using the optimized CPA-LFD method for *Laenia aquaticis* in Examples 2 and 3, with a blank control included. The results are as follows: Figure 4 As shown in Figures 1-8, the DNA concentrations of *Laenia aquaticis* were 48 ng, 4.8 ng, 480 pg, 48 pg, 4.8 pg, 480 fg, 48 fg, and 4.8 fg / ul, respectively; Figure 9 shows the negative control. The results indicate that the CPA-LFD method of this invention has a minimum detection limit of 48 fg / ul for *Laenia aquaticis*, demonstrating high sensitivity.
[0032] Example 9: Rapid detection of clinical samples using the CPA-LFD method and its kit application. The CPA-LFD method 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, forekera, somatic kidney, foregut, midgut, hindgut, and gills of the crucian carp using a bacterial genomic DNA extraction kit. Then, the CPA-LFD reaction was performed using the reaction system and conditions described in Example 2. After the PCR reaction was completed, 10 μL of the PCR product was used for detection on a nucleic acid test strip. The results are as follows: Figure 5 As shown, 1-8: liver, spleen, anterior kidney, somatic kidney, foregut, midgut, hindgut, gills; 9: negative control. The results indicate that all clinical samples were positive, and the negative control was negative, demonstrating that the CPA-LFD method of this invention can be used for the detection of clinical samples.
[0033] 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 for detecting *Laenia aquaticis*, characterized in that, The primer set includes two DNA probe primers based on single-cross primer isothermal amplification (CPA) technology, namely P1 and P2, with sequences shown in SEQ ID NO.1~2: P1: 5'-Biotin-CAACGGTGCTAATGGTCG-3' (SEQ ID NO.1) P2: 5'-FAM-CGTAAGTGATCATGACACTG-3' (SEQ ID NO. 2).
2. The specific primer sequence as described in claim 1, characterized in that, The 5' end of primer P1 is labeled with biotin, and the 5' end of primer P2 is labeled with 6-carboxyfluorescein (6-FAM). Therefore, the amplification product of CPA contains both FAM and biotin labels.
3. The application of the specific primer sequences as described in claims 1 and 2 in the preparation of reagents or kits for the detection of aquatic Ranuncula using the CPA-LFD method.
4. The application as described in claim 3, characterized in that, The kit also includes dNTPs, Bst DNA polymerase, Thermopol Buffer, MgSO4, betaine, etc.
5. The application as described in claim 3, characterized in that, The kit also includes nucleic acid detection test strips, which are colloidal gold transverse flow test strips (LFD).
6. The application as described in claim 3, characterized in that, The CPA system of the reagent or kit is as follows: 20 μL reaction volume: 2 μL 10×Thermopol Buffer; 0.2 μL each of forward and reverse primers 5F and 3R; 1.2 μL single cross primer PF; 0.6 μL CPA probe primer P1; 0.6 μL CPA probe primer P2; 1 μL Bst DNA polymerase; 0.6 μL dNTPs; 8 μL betaine; 0.2 μL MgSO4; 3 μL DNA; and ddH2O to make up to 20 μL.
7. The application as described in claim 3, characterized in that, The CPA reaction conditions for the reagent or kit are as follows: react at 63°C for 45-60 min, and then inactivate at 80°C for 5 min.
8. The nucleic acid detection test strip as described in claim 5, characterized in that: The test strip measures 1cm x 10cm and has two sides. From the front, the layers from bottom to top are: sample pad, conjugation pad, interpretation area, and absorption pad. The interpretation area includes a test line (T) and a control line (C). Anti-Biotin antibody is labeled on the control line, and anti-FAM antibody is labeled on the test line. When the CPA amplification final product labeled with FAM and Biotin is added to the test strip, it binds to the colloidal gold containing the anti-Biotin antibody. This binds to the anti-FAM antibody at the test line, resulting in a visible red line. If there is no double-labeled amplification product, the colloidal gold bound to the anti-Biotin antibody will not be captured by the anti-FAM antibody, and no red line will be visible on the test line. In addition, the nucleic acid test strip is single-sided, with only the front side showing color and the back side not showing color. After adding 5-10 μL of CPA amplification final product to the test strip, add 100 μL of buffer solution, and the results can be observed within 5-10 minutes.
9. The application as described in claim 3, characterized in that, The steps include: (1) Configuring the CPA reaction system: The template is the DNA of the sample to be tested; (2) CPA reaction: The CPA reaction system in step (1) is reacted at 63°C for 45-60 min, and then inactivated at 80°C for 5 min. The amplified product is detected by nucleic acid test strips, and the results are observed within 5-10 min. (3) Result interpretation, red light method observation: 1) Positive (+): Red lines appear in both the control area and the test area, and a total of two red lines appear on the test strip; 2) Negative (-): One red line appears in the control area of the test strip, and there is no band in the test area; 3) Invalid: No red lines appear in either the control area or the test area of the test strip.