Rapid typing detection method for two pathogens of penaeus vannamei by naked eye visual RPA-PfAgo method
High-distinguishing molecular beacons were designed through naked-eye visualization RPA-PfAgo method, and specific cutting was used for PfAgo, which solved the problem of rapid detection of WSSV and AHPND in South American white shrimp farming, and achieved high-sensitivity multiple pathogen detection, which was suitable for on-site detection of aquaculture.
Patent Information
- Application Number
- CN202510568352.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-15
AI Technical Summary
The existing nucleic acid detection methods cannot effectively and rapidly detect leukoplakia syndrome virus (WSSV) and acute hepatopancreatic necrosis (AHPND) in the breeding of white shrimps in South America, and the existing isothermal amplification technology has insufficient sensitivity and credibility, so it is impossible to achieve simultaneous typing detection of multiple pathogens.
The naked-eye visualization RPA-PfAgo method was used to design two high-distinguishing molecular beacons, and three gDNA-guided PfAgo for specific cleavage was used to achieve a reaction to detect multiple pathogens, and different detection results were indicated by the combination of fluorescent signals.
It realizes high sensitivity detection of WSSV and AHPND in on-site detection scenarios, and can observe different results through the naked eye, expanding the application field of PfAgo in aquatic pathogen detection.
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Figure CN120485432A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of aquatic disease gene typing detection, and in particular to a method for rapid typing detection of two pathogens of whiteleg shrimp using a naked-eye visualized RPA-PfAgo method. Background Art
[0002] White spot syndrome virus (WSSV) and acute hepatopancreatic necrosis disease (AHPND) are two important pathogens in whiteleg shrimp aquaculture. Once infected, they can rapidly cause widespread infection and mortality throughout the shrimp pond. The current lack of effective treatments for these two diseases results in significant annual economic losses for the shrimp aquaculture industry. Therefore, early detection of WSSV and AHPND in whiteleg shrimp aquaculture is crucial for the prevention and control of these two diseases. PCR-based nucleic acid detection methods are the most commonly used for early detection of WSSV and AHPND. However, these methods rely on sophisticated thermal cycling equipment, making them unsuitable for resource-limited field testing. With the development of isothermal amplification technology, a large number of rapid detection methods based on isothermal amplification have emerged, such as RPA and LAMP. These methods avoid the need for thermal cycling equipment and hold great potential for on-site testing. However, relying solely on amplification for signal amplification and readout presents limitations in sensitivity and reliability. In recent years, detection technologies combining isothermal amplification with nucleases have been widely used in nucleic acid detection. For example, the currently popular SHARLOCK technology is a rapid detection method developed by combining RPA technology with CRISPR / Cas12a nuclease, but this method cannot perform one-tube detection of multiple pathogens. Pf Ago is a programmable nuclease that can specifically recognize target sequences and cut at specific sites under the guidance of single-stranded DNA. Pf Ago enzymes are derived from thermophilic bacteria Pyrocococcus furiosus Argonaute has good thermal stability and can be transported and stored at room temperature; the selection of targets does not require consideration of the PAM sequence, which also shows that its application convenience is better than that of the CRISPR series nucleases. Pf This ability of Ago to specifically cut molecular beacons, which is different from Cas12a, gives it a natural advantage in multiplex detection. For example, in 2024, Yixin Tang et al. established a dual RPA- PfThe Ago method, applied to genotyping of LZTFL1 allele mutation sites, achieved single-copy sensitivity. However, this method was unable to differentiate mixed double samples using fluorescence visualization within a single tube. Therefore, an optimization approach was developed using two high-resolution molecular beacons designed based on optical principles to enable simultaneous genotyping of WSSV and AHPND in a single tube. Summary of the Invention
[0003] The present invention is based on RPA- Pf Ago dual detection technology uses two highly differentiated molecular beacons designed based on optical principles and three cleverly designed gDNA guides Pf Ago performs precise and specific cutting to achieve the purpose of detecting multiple pathogens in one reaction. A naked eye visualization RPA-PfAgo method was established for the rapid typing of two pathogens in white shrimp. This method only requires simple equipment support, and the fluorescence output results can be directly observed by the naked eye, which has good application potential in on-site detection scenarios. This study is also the first to use Pf Ago enables simultaneous typing and detection of two important aquaculture pathogens. This dual detection method is also applicable to the development of multiple on-site detection methods for other important pathogens in aquaculture, which is of great value in promoting the healthy farming of whiteleg shrimp. The specific technical solution is as follows: A naked-eye visualized RPA-PfAgo method is used for rapid typing and detection of two pathogens of white shrimp. The technical scheme is as follows: the detection includes detection of white spot syndrome virus and / or acute hepatopancreatic necrosis disease, and the detection includes a designed primer set, three gDNAs and a molecular beacon.
[0004] Further, the primer set includes WSSV-F, WSSV-R, AHPND-F and AHPND-R, and their gene sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The three gDNAs include gDNA1-WSSV, gDNA2-AHPND, and gDNA3-AHPND, and their gene sequences are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively. The molecular beacons include MB-WSSV and MB-AHPND, and their gene sequences are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.
[0005] Furthermore, the detection method comprises the following steps: (1) Preparation of bacterial strains and diseased shrimp genomic DNA; (2) PfExpression and purification of Ago; (3) Standard plasmids for WSSV and AHPND; (4) Duplex RPA amplification reaction system; (5) Visualized fluorescence dual RPA- Pf Ago reaction system.
[0006] Furthermore, the duplex RPA amplification reaction system is specifically prepared by adding the following to a tube of lyophilized powder: 25 μL of buffer A; 2 μL each of 20 μM RPA primers WSSV-F, WSSV-R, AHPND-F, and AHPND-R; 3 μL of WSSV template DNA; 5 μL of AHPND template DNA; 6.5 μL of deionized water; and 2.5 μL of buffer B; and initiating the reaction at 37° C. for 30 minutes.
[0007] Furthermore, the visualized fluorescence dual RPA- Pf The Ago reaction system is based on a 20 μL system, including: 10 μL of purified AHPND and WSSV dual RPA amplification products; 2.5 μM Pf Ago; 1 μM (final concentration) gDNA1-WSSV; 0.5 μM (final concentration) gDNA2-AHPND; 0.5 μM (final concentration) gDNA3-AHPND; 1 μM (final concentration) MB-AHPND; 0.6 μM (final concentration) MB-WSSV; and 2 μL of 10× reaction buffer (2.5 M NaCl, 5 mM MnCl2, 200 mM HEPES, pH 7.5); use deionized water to make up the total volume to 20 μL; incubate at 95°C for 60 min, recording FAM and Texas Red fluorescence intensities every 1 min. The endpoint fluorescence signal is obtained by placing the reaction tube under blue light after the reaction is completed and observing with the naked eye. The fluorescence signal of the reaction tube is photographed using a smartphone. Furthermore, the Pf Ago expression and purification include pET-28a- Pf Ago plasmid construction, Pf Ago protein expression and purification pretreatment and Pf Purification of Ago protein.
[0008] The above technical solution can achieve the following beneficial effects: Compared with the existing technology, the visual dual RPA- PfThe convenience of the Ago method is reflected in the following aspects: the entire reaction does not require precise thermal cycling equipment and can be completed within 1.6 hours. Through the comprehensive design of the single-tube fluorescent signal combination, color indication of different test results is achieved, that is, different results can be observed with the naked eye. The present invention provides a highly sensitive on-site detection method for the prevention and control of WSSV and AHPND in shrimp farming. Pf Ago has achieved the simultaneous typing detection of multiple aquatic pathogens for the first time, expanding Pf Ago's application areas are worth promoting. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 Is to visualize fluorescence duplex RPA- Pf Schematic diagram of the Ago method.
[0010] Figure 2 Is to visualize fluorescence duplex RPA- Pf Ago sensitivity.
[0011] Figure 3 Specificity validation was performed using a panel of pathogen genomes associated with shrimp aquaculture. DETAILED DESCRIPTION
[0012] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative work are within the scope of protection of the present invention.
[0013] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.
[0014] The present invention is described in detail below with reference to the accompanying drawings to facilitate those skilled in the art to understand the present invention.
[0015] 1. Reagents and Instruments Bacterial genomic DNA extraction kit, marine animal tissue genomic DNA extraction kit, and plasmid mini-extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; RPA nucleic acid amplification kit was purchased from Hangzhou Zhongce Biotechnology Co., Ltd.; 2×Taq Master Mix (Dye Plus), 2×Phanta Max Master Mix (Dye Plus), and FastPure Gel DNA Extraction Mini Kit were purchased from Nanjing Novozymes Biotechnology Co., Ltd.; MiniProEpBasic basic electrophoresis instrument was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; GelDoc Go gel imaging system was purchased from Bio-Rad Corporation, USA; pMD-18 T vector was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; low-temperature ultrahigh pressure homogenizer was purchased from Shanghai Hongli Biotechnology Co., Ltd.; Nanodrop Lite spectrophotometer was purchased from Thermo Fisher Scientific (China) Co., Ltd.; and Roche LightCycler 480 II qPCR instrument was purchased from Switzerland. To prepare the Lysis Buffer protein resuspension buffer: Add 15 mL of 1M Tris-HCl (pH 8.0) solution, 56.25 mL of 4M NaCl solution, and 1.5 mL of 1M MnCl2 solution to a beaker. Add water to a total volume of 750 mL. Filter the solution and place in a 4°C display cabinet until ready for use. To prepare the Elute Buffer protein elution buffer: Add 6 mL of 1M Tris-HCl (pH 8.0) solution, 22.5 mL of 4M NaCl solution, 0.6 mL of 1M MnCl2 solution, and 24 mL of 1M imidazole solution to a beaker. Add water to a total volume of 300 mL. Filter the solution and place in a 4°C display cabinet until ready for use. To prepare the Storage Buffer protein dialysate: Add 20 mL of 1M Tris-HCl solution, 75 mL of 4M sodium chloride solution, 0.5 mL of 1M manganese chloride solution to a beaker. Finally, add 150 mL of glycerol, mix thoroughly, and place in a 4°C display cabinet until ready for use. Example
[0016] As attached Figure 1-3As shown, a naked eye visualized RPA-PfAgo method is used for rapid typing detection of two pathogens of white shrimp, the detection including detection of white spot syndrome virus and / or acute hepatopancreatic necrosis disease, and the detection method includes a designed primer set, three gDNAs and a molecular beacon; the primer set includes WSSV-F, WSSV-R, AHPND-F and AHPND-R, whose gene sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively; the three gDNAs include gDNA1-WSSV, gDNA2-AHPND, and gDNA3-AHPND, whose gene sequences are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively; the molecular beacons include MB-WSSV and MB-AHPND, whose gene sequences are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively; the above sequence table is shown in Table 1:
[0017] Table 1 Gene sequence list As a preferred embodiment, the detection method comprises the following steps: (1) Preparation of bacterial strains and diseased shrimp genomic DNA Diseased shrimp samples (WSSV, SHIV, and AHPND) for this experiment were provided by the Jiangsu Institute of Marine Fisheries, and CQIV samples were provided by Zhang Xiaojun's research group at Yangzhou University. All samples were validated by qPCR. Shrimp tissue was ground into a homogenate using a grinder and sterilized by UV irradiation for 30 minutes. DNA was then extracted from the homogenate using a marine animal tissue genomic DNA extraction kit (see the instructions for specific procedures).
[0018] The bacterial samples used in this experiment, including Aeromonas hydrophila, Edwardsiella tarda, Listeria monocytogenes, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio harveyi, Vibrio cholerae, and Vibrio alginolyticus, were inoculated on alkaline peptone solid medium and cultured overnight at 37°C. Ten single clones were selected and placed in alkaline peptone water medium and cultured until OD 600 to 0.8; finally, follow the instructions of the bacterial genomic DNA extraction kit.
[0019] (2) Pf Ago expression and purification Step 1, pET-28a- Pf Ago plasmid construction: Obtained from previously reported literature PfThe gene sequence of Ago was synthesized by General Biotechnology (Anhui) Co., Ltd. Two restriction enzyme cloning sites, NdeI and XhoI, were selected. Pf The Ago gene was cloned into the pET28b(+) vector to construct the pET28b-6×His- Pf Ago.
[0020] Step 2 Pf Ago protein expression and pre-purification treatment: 1) Transform the recombinant plasmid into E. coli BL21 (DE3) pLysS expression competent cells, inoculate onto LB plates containing 50 µg / mL (final concentration) Kana resistance and 35 µg / mL (final concentration) Chl resistance, and culture overnight at 37°C. 2) Pick 10 single colonies and incubate in 20 mL LB liquid medium containing 50 µg / mL (final concentration) Kana resistance and 35 µg / mL (final concentration) Chl resistance. Incubate at 37°C, 220 rpm for 3-4 hours. 3) At a ratio of 1:100, take 5 mL of activated bacterial solution and add it to 500 mL of LB medium containing 50 µg / mL (final concentration) of Kana resistance and 35 µg / mL (final concentration) of Chl resistance. Incubate at 37°C, 220 rpm for 5-6 h (OD 600 is 0.6-0.8); 4) Collect 1 mL of bacterial culture as a pre-induction sample and store it in a 4°C display cabinet for protein gel preparation. Add 0.5 mL of IPTG (1 M) to 500 mL of expansion medium to induce target protein expression. Induce overnight at 16°C, 220 rpm for 12 h. Collect 1 mL of bacterial culture as a post-induction sample and store it in a 4°C display cabinet for protein gel preparation. Collect the bacterial pellet using a high-speed refrigerated centrifuge at 4°C, 8000 rpm, for 10 min, and discard the supernatant LB medium. Store the collected bacterial pellet at -80°C overnight. 5) Weigh the collected cells and add Lysis Buffer (10 mL: 1 g) to the cell weight. Vortex until the cell suspension is dissolved and no clumps of cell pellet remain. Then, use a 20 mL sterile syringe to draw up the cell solution and insert a 5 mL sterile syringe needle into the syringe to repeatedly pipette and aspirate to further dissolve the cells (add protease inhibitors if necessary). 6) Use a low-temperature ultrahigh-pressure homogenizer to disrupt the resuspended bacteria. Before use, connect the low-temperature ultrahigh-pressure homogenizer to a power source, turn on the condenser, set it to 4°C, and start the circulating fan. Empty the sealed 20% alcohol in the sample cup and rinse the tubing with ultrapure water. Repeat this rinse for at least three sample cups. Then, rinse the tubing with at least 50 mL of Lysis buffer. Use the syringe mentioned in the previous step to aspirate the resuspended bacteria, keeping the needle close to the wall of the sample cup and slowly inject the sample to avoid bubbles that may cause clogging. Disrupt the mixture at 85 MPa and 4°C for 15 minutes. The bacterial solution will change from a viscous white to a clear, translucent, and yellowish color. 7) Centrifuge the crushed and collected samples at 16,000 rpm in a high-speed refrigerated centrifuge at 4°C for 45 minutes. Collect the supernatant and dissolve the precipitate as the insoluble component in 50 mL of PBS buffer and store in a 4°C display cabinet for protein gel preparation. 8) Heat the crude enzyme solution in a water bath at 80°C for 30 minutes. Centrifuge again at 16,000 rpm at 4°C for 45 minutes in a high-speed refrigerated centrifuge. Collect the supernatant and discard the creamy white protein precipitate. Place the supernatant on ice and filter through a 0.22 µM filter into a 50 mL centrifuge tube. Take 1 mL of the supernatant as the supernatant sample and store it in a 4°C display cabinet for protein gel preparation. Prepare the remaining supernatant for purification on the AKTA instrument.
[0021] Step 3 Pf Purification of Ago protein: 1) Remove pumps A and B from the 20% alcohol, rinse with a wash bottle, and place in ultrapure water. Set the AKTA Primeplus system's built-in program to perform System Wash. After stopping the wash, install a 1 mL NTA-Ni prepacked column and set Manual Run to adjust the flow rate to 1 mL / min and the pressure to 0.3 MPa to flush 10 column volumes. 2) Place pump A in lysis buffer and pump B in elution buffer. Set the AKTA Primeplus system program to perform System Wash. After stopping the wash, set a Manual Run with a flow rate of 1 mL / min and a pressure of 0.3 MPa to wash the nickel column for 10 minutes to equilibrate it. 3) Place pump A in the supernatant, set the flow rate to 1 mL / min, and adjust the pressure to 0.3 MPa for sample loading. After sample loading, rinse pump A and place it in lysis buffer. Set the flow rate to 1 mL / min and adjust the pressure to 0.3 MPa. After rinsing, connect 1 mL of flow to the waste outlet of the machine as the gel sample and store it in a 4°C display cabinet for protein gel preparation. 4) Set up a Manual Run with a flow rate of 1 mL / min, pressure adjusted to 0.3 MPa, and a concentration of 5% B%. Perform isocratic elution (30 min) of non-target impurities remaining on the nickel column with 5% low-concentration imidazole. Observe the UV absorption curve on the computer. When a peak appears, collect 1 mL of the eluate as a gel wash sample and store it in a 4°C display cabinet for protein gel preparation. End the 5% isocratic elution procedure when the curve stabilizes. 5) Set up a Manual Run using an automated collector, 1 mL / tube, and gradient elution conditions: Length 8 mL; Target 100%, flow rate 1 mL / min, pressure adjusted to 0.3 MPa, and Concentration B % 100% (the Elute Buffer percentage in pump B increases from 5% to 100% over 8 minutes). Observe the UV absorbance curve and mark the collection tube where the peak appears. Elute for 20 minutes, ending the run when the imidazole peak on the curve stabilizes. 6) Rinse pumps A and B and place them in ddH2O. Set the AKTA Prime plus system's built-in program to perform System Wash. After stopping the automatic wash, set Manual Run to adjust the flow rate to 1 mL / min and the pressure to 0.3 MPa to flush 10 column volumes. 7) After cleaning pumps A and B, place them in 20% ethanol and set the AKTA Prime Plus system's built-in program to perform System Wash. After stopping the automatic wash, set the flow rate to 1 mL / min and the pressure to 0.3 MPa to flush for 2-3 column volumes. Then, remove the nickel column and store it in a display cabinet at 4°C. 8) Analyze the purified eluate using 10% SDS-PAGE gel electrophoresis. Collect 40 µL each of the pre-induction, post-induction, flow, and wash samples from a 4°C display cabinet, add 10 µL of 5× protein loading buffer, and vortex centrifuge. Heat at 100°C for 10 minutes, then vortex centrifuge again. Load 20 µL of sample and run the gel using the following conditions: upper gel at 90 V for 30 minutes, lower gel at 150 V for 1 hour. After electrophoresis, microwave-stain the gel using Coomassie Brilliant Blue R250 staining solution at medium heat for 5 minutes. After staining, microwave-decolorize the gel using pure water at medium-high heat for 10-15 minutes. Finally, visualize the gel using a Bio-Rad gel imaging system. 9) Mix the high-concentration and low-concentration protein samples separately based on the protein gel image and dialyze them overnight using Storage Buffer (prepared and used immediately). The next day, re-prepare the Storage Buffer and replace the old Storage Buffer for a second dialysis. The dialysis time should be at least 2 hours. 10) Prepare 0.25mg / mL, 0.5mg / mL, 0.75mg / mL, 1mg / mL, and 1.25mg / mL BSA protein; take BSA protein solutions of different concentrations and the dialyzed Pf 40µL of each Ago protein was added, followed by 10µL of 5× protein loading buffer and vortex centrifugation. After heating at 100°C for 10 minutes, vortex centrifugation was performed again. 20µL of sample was loaded for gel electrophoresis. The grayscale value of the gel image was used to estimate the dialyzed Pf The concentration of Ago protein was determined; finally, the purified protein containing glycerol was aliquoted into small volumes (20 µL / tube) and stored in a -80°C refrigerator.
[0022] (3) Standard plasmids for WSSV and AHPND Step 1: Construction of WSSV standard plasmid: Firstly, genomic DNA was extracted from shrimp infected with WSSV, and the vp28 gene fragment (GenBank accession no. AF 173993) with high specificity on the WSSV gene was selected as the target gene. Primers WSSV-757 FP and WSSV-757 RP were used to amplify the target gene fragment (757 bp) by PCR and cloned into the pMD18-T vector via TA.
[0023] The specific operation of TA cloning is as follows: 1) Amplify the target fragment using the high-fidelity enzyme 2×Phanta Max Master Mix (Dye Plus); 2) Prepare a 1% agarose gel, load all the amplified products according to the volume of the gel pores, and then perform electrophoresis at 90V for 40 minutes. The amplified products can be preliminarily confirmed by comparing the size of the marker bands on the gel image with the target fragments; transfer the agarose gel to a blue light gel cutter and cut the agarose gel containing the target DNA under blue light irradiation; use a sterile blade and disinfect it with 70% ethanol and a nucleic acid remover before cutting to reduce the risk of contamination; weigh the cut gel mass and calculate the volume of buffer to be added according to the ratio of 1mg=1µL; then heat it at 65℃ for 10-15min, shaking it upside down every 5 minutes until the gel is completely melted; transfer the dissolved gel solution to an adsorption column and centrifuge to remove impurities; then wash it multiple times with WashBuffer according to the Fast Pure Gel DNA Extraction Mini Kit to remove salt and other impurities; finally, use Elution Buffer preheated at 55℃ or deionized water to wash the DNA off the core column; 3) Prepare the DNA reaction solution by placing 1 µL of 50 ng / µL pMD18-T vector, 1 µL of 50 ng / µL WSSV-DNA insert, and 1 µL of deionized water in a 0.2 mL sterile centrifuge tube. 4) Add 5 µL (equal volume) of Ligation Solution I and react at 16°C for 30 minutes (overnight reaction does not affect efficiency). Transform the entire amount (10 µL) into 100 µL of DH5α competent cells. Cultivate on LB agar plates containing Amp to form single colonies. Select white colonies and use colony PCR to confirm the length of the insert in the T vector, which also further confirms the accuracy of the target sequence. 5) Sequencing (Anhui General Biotechnology Co., Ltd.) was performed and compared with the sequence published in Genbank, and quantified using Nanodrop. The copy number was calculated based on the size of the recombinant plasmid (3451 bp). A standard curve was constructed after 10-fold dilution to show the correlation between Ct and copy number. The correlation between copy number and Ct was calculated based on the qPCR results as Ct = −3.710 * lg (DNA copy number) + 36.49, R 2 =0.9994.
[0024] Step 2: Construction of AHPND standard plasmid: The AHPND plasmid is a natural plasmid pVPA3-1 extracted from shrimp infected with Vibrio parahaemolyticus AHPND; The copy number of DNA standards was determined by qPCR; briefly, the virulence plasmid pVPA3-1 was extracted using the TIANprep MiniPlasmid Kit (TIANGEN Biotech Co Ltd, Beijing, China) and quantified using a Nanodrop ultraviolet spectrophotometer.
[0025] Step 3: Duplex RPA amplification reaction system To one tube of lyophilized powder, add 25 μL of Buffer A, 2 μL each of 20 μM RPA primers (WSSV-F, WSSV-R, AHPND-F, and AHPND-R), 3 μL of WSSV template DNA, 5 μL of AHPND template DNA, and 6.5 μL of deionized water. Finally, add 2.5 μL of Buffer B to initiate the reaction. Incubate the reaction at 37°C for 30 min.
[0026] Example 1: Visualization of Fluorescence Duplex RPA- Pf Ago reaction system Pf The total Ago reaction system was 20 μL, including 10 μL of purified AHPND and WSSV dual RPA amplification products, with a final concentration of 2.5 μM. Pf Ago, 1 μM gDNA1-WSSV, 0.5 μM gDNA2-AHPND, 0.5 μM gDNA3-AHPND, 1 μM MB-AHPND, 0.6 μM MB-WSSV, and 2 μL of 10× reaction buffer (2.5 M NaCl, 5 mM MnCl2, 200 mM HEPES, pH 7.5). The reaction was performed on a Roche LightCycler 480 II qPCR instrument (Basel, Switzerland) at 95°C for 60 min. FAM and Texas Red fluorescence intensities were recorded every 1 min. Endpoint fluorescence was determined by visually observing the reaction tubes under blue light after the reaction was complete and capturing the fluorescence signal using a smartphone.
[0027] Test results such as Figure 1 As shown, when the target sequence of WSSV is present, the molecular beacon of WSSV is cut and a green fluorescence signal is generated under a blue light source; when the target sequence of AHPND is present, the molecular beacon of AHPND is cut and a red fluorescence signal is generated under a blue light source; when both the target sequence of WSSV and the target sequence of AHPND are present, both the molecular beacon of WSSV and the molecular beacon of AHPND are cut and a yellow fluorescence signal is generated under a blue light source; when neither the target sequence of WSSV nor the target sequence of AHPND is present, neither the molecular beacon of WSSV nor the molecular beacon of AHPND is cut and no fluorescence signal is generated under a blue light source.
[0028] Example 2: Visualization of Fluorescence Duplex RPA- Pf Sensitivity of the Ago method The dual RPA- Pf The sensitivity of the Ago method was evaluated. Figure 2 As shown, (AB) visualizes the fluorescent doublet RPA- Pf Sensitivity of Ago to single target: Real-time fluorescence curve and end-point image display method for 10 0 -10 4 Detection results of WSSV (A) or AHPND (B) standard DNA copies; (C) Visualized fluorescence duplex RPA- Pf The sensitivity of Ago to dual targets: the concentration of 10 0 -10 4 WSSV and AHPND standard DNA copies were mixed at a 1:1 ratio as templates for the reaction; the detection results were presented as endpoint signals and images.
[0029] Fluorescence curve results showed that the dual RPA- Pf The sensitivity of the Ago method for detecting WSSV can reach 10 0 Copy per reaction ( Figure 2 A) The sensitivity of detecting AHPND is 10 2 Copy per reaction ( Figure 2 B), the end-point fluorescence results are consistent with the fluorescence curve results ( Figure 2 C). The detection sensitivity of this method for both pathogens was higher than or comparable to the current gold standard method.
[0030] Example 3: Visualization of Fluorescence Duplex RPA- Pf Specificity of the Ago method Dual RPA was validated using a panel of shrimp aquaculture-related pathogens including SHIV, EHP, V. parahaemolyticus, V. vulnificus, V. harveyi, V. alginolyticus, and V. cholerae. Pf The specificity of the Ago method. The results showed that fluorescence signals were observed only when WSSV and / or AHPND targets were added to the reaction system; when only other samples were added to the reaction system, no fluorescence signals were generated. This shows that the method has good specificity. Figure 3 As shown, the results are presented as endpoint fluorescence signal histograms and images.
[0031] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention are within the scope of protection of the claims attached to this application.
Claims
1. A method for rapid typing and detection of two pathogens of whiteleg shrimp using naked-eye visualization RPA-PfAgo, characterized by: The detection includes the detection of white spot syndrome virus and / or acute hepatopancreatic necrosis disease, and the detection method includes a designed primer set, three gDNAs and a molecular beacon.
2. The method according to claim 1, wherein the naked eye visualization RPA-PfAgo method is used for rapid typing detection of two pathogens of whiteleg shrimp, characterized in that: The primer set includes WSSV-F, WSSV-R, AHPND-F and AHPND-R, and the gene sequences thereof are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively; The three gDNAs include gDNA1-WSSV, gDNA2-AHPND, and gDNA3-AHPND, and their gene sequences are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively; The molecular beacons include MB-WSSV and MB-AHPND, and their gene sequences are shown in SEQ ID NO.8 and SEQ ID NO.9 respectively.
3. The method for rapid typing detection of two pathogens of whiteleg shrimp using a naked eye visualization RPA-PfAgo method according to claim 1 or 2, characterized in that: The steps include: (1) Preparation of bacterial strains and diseased shrimp genomic DNA; (2) Pf Expression and purification of Ago; (3) Standard plasmids for WSSV and AHPND; (4) Duplex RPA amplification reaction system; (5) Visualized fluorescence dual RPA- Pf Ago reaction system.
4. The method according to claim 3, wherein the naked eye visualization RPA-PfAgo method is used for rapid typing and detection of two pathogens of Penaeus vannamei, characterized in that: The duplex RPA amplification reaction system specifically includes adding 25 μL of buffer A; 2 μL each of 20 μM RPA primers WSSV-F, WSSV-R, AHPND-F, and AHPND-R; 3 μL of WSSV template DNA; 5 μL of AHPND template DNA; 6.5 μL of deionized water; and 2.5 μL of buffer B to a tube of lyophilized powder. The reaction is started at 37°C for 30 minutes.
5. The method according to claim 3, wherein the naked eye visualization RPA-PfAgo method is used for rapid typing and detection of two pathogens of Penaeus vannamei, characterized in that: The visualized fluorescent dual RPA- Pf The Ago reaction system is specifically based on 20 μL, including 10 μL of purified AHPND and WSSV double RPA amplification products; 2.5 μM Pf Ago;1μM gDNA1-WSSV;0.5μM gDNA2-AHPND; 0.5μM gDNA3-AHPND; 1μM MB-AHPND; 0.6 μM MB-WSSV; 2 μL 10× reaction buffer (2.5 M NaCl, 5 mM MnCl2, 200 mM HEPES, pH 7.5); add deionized water to the total volume of 20 μL; incubate at 95°C for 60 min, with FAM and Texas Red fluorescence intensities recorded every 1 min. The endpoint fluorescence signal was obtained by placing the reaction tube under blue light and observing with the naked eye after the reaction. The fluorescence signal of the reaction tube was photographed using a smartphone.
6. A method for rapid typing and detection of two pathogens of whiteleg shrimp using a naked eye visualization RPA-PfAgo method according to claim 3, characterized in that: described Pf Ago expression and purification include pET-28a- Pf Ago plasmid construction, Pf Ago protein expression and purification pretreatment and Pf Purification of Ago protein.