Combined detection method for escherichia coli O157 and salmonella typhimurium based on RPA-Exo multiple fluorescence
Through RPA-Exo multiple fluorescence technology, Exonuclease III nuclease is used to cut the fluorescent probe during the RPA amplification process, which solves the problems of long cycle, high false positive rate and equipment complexity of traditional detection methods, and realizes fast and simple dual nucleic acid detection, which is suitable for food safety testing.
Patent Information
- Application Number
- CN202510568354.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-09-12
AI Technical Summary
Existing technologies have problems such as long cycles, complex equipment, high false positive rates and high costs when detecting Escherichia coli O157 and Salmonella typhimurium. Traditional methods are difficult to meet the needs of primary clinics, disaster sites or home self-testing.
Using RPA-Exo multiplex fluorescence technology, two high-resolution molecular beacons were designed. Exonuclease III exonuclease was used to specifically cleave the fluorescent probe during the RPA amplification process, generating different fluorescent signals and achieving multiple nucleic acid detection in a single tube.
It realizes fast, simple and visual dual nucleic acid detection in a single tube with high sensitivity, which is suitable for food safety emergencies and routine testing, reducing the complexity of equipment and operations.
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Figure CN120624686A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of foodborne pathogen detection, and in particular to a combined detection method for Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence. Background Art
[0002] Shiga toxin-producing Escherichia coli (STEC) infections account for 20% of all foodborne illnesses worldwide, with E. coli O157 being the most representative. Salmonella typhimurium is the most common serotype of Salmonella isolated from food samples, and its incidence rate ranks first among Salmonella species. Current detection technologies for these two foodborne pathogens primarily encompass three approaches: traditional isolation and identification methods based on microbial culture, immunoassays based on antigen-antibody reactions, and molecular diagnostics based on nucleic acid sequence analysis. However, traditional isolation and identification methods require specialized microbiology laboratories, and a complete identification cycle typically takes 5-7 working days, resulting in lengthy and complex technical processes. Immunoassays based on antigen-antibody specific reactions are epitope-dependent, potentially generating false negatives when the target epitope mutates. Furthermore, the high costs associated with antibody preparation and purification have limited the widespread adoption of these methods. Modern molecular diagnostic techniques primarily include polymerase chain reaction (PCR) and isothermal amplification. Isothermal amplification, a recently developed technique for nucleic acid amplification at a constant temperature, is a type of amplification, such as RPA and LAMP. This technique does not require a thermal cycler, and the equipment can be simplified to a handheld format, simplifying the operation process and eliminating the need for specialized personnel. It is suitable for use in primary care clinics, disaster sites, or for home testing. Traditional nucleic acid amplification results rely on agarose gel electrophoresis, which is susceptible to aerosol contamination and can lead to false-positive results in subsequent experiments. In contrast, visual inspection methods can effectively prevent laboratory aerosol contamination, avoid false-positive results in subsequent experiments, save equipment and time required for electrophoresis, and avoid the risk of toxicity during the electrophoresis process. Combining isothermal amplification with nucleases, leveraging the specificity of nuclease cleavage of molecular beacons to produce distinct fluorescent signals, is currently an effective strategy for visual multiplex nucleic acid detection. Therefore, based on this, research was conducted using two high-resolution molecular beacons designed according to optical principles. On the basis of RPA isothermal amplification, the nuclease Exonuclease III and the Exo fluorescent probe were added. During the RPA amplification process, the Exo probe specifically binds to the two targets to form double-stranded structures. Exonuclease III will specifically cut the fluorescent probe (THF site), causing the fluorescent group to separate from the quenching group, generating different fluorescent signals, so as to achieve simultaneous genetic detection of two important foodborne pathogens, Escherichia coli O157 and Salmonella typhimurium, in a single tube. Summary of the Invention
[0003] This paper develops a multi-fluorescence RPA-Exo method for the simultaneous and rapid detection of two important foodborne pathogens, Escherichia coli O157 and Salmonella typhimurium. This method requires only simple equipment, and the fluorescence output can be directly observed by the naked eye, showing great potential for on-site detection. The specific technical solution is as follows: A combined detection method for Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence, the technical scheme of which is as follows: the detection method designs two high-discrimination molecular beacons based on optical principles, and uses two cleverly designed probes for precise specific cutting to achieve the purpose of detecting Escherichia coli O157 and / or Salmonella typhimurium in one reaction. The probes include O157-Exo probe, rfb The E-Exo probe and its corresponding probe complementary long primers are O157-Exo probe complementary primer, rfb The complementary primers of E-Exo probe, whose gene sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively; the primers include O157-F, O157-R, rfb EF, rfb ER, whose gene sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.
[0004] Furthermore, a combined detection method for Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence comprises the following steps: (1) Preparation of double-stranded Exo fluorescent probe standards; (2) Preparation of plasmid standards; (3) Preparation of bacterial liquid nucleic acid standards; (4) Application of dual fluorescence RPA-Exo reaction system; (5) Generate fluorescence by blue light irradiation and produce a fluorescence signal graph.
[0005] Furthermore, the application of the dual fluorescence RPA-Exo reaction system includes the detection of Escherichia coli O157 and / or Salmonella typhimurium, with a volume of 50 μL, and the specific steps are as follows: 10µM primers O157-F, O157-R, rfb EF, rfb 1 µL each for ER; 10µM O157-Exo probe 0.3µL, rfb E-Exo probe 0.3µL; A Buffer 25µL; ddH2O 9.9µL; B Buffer 2.5µL; 4 µL of Escherichia coli O157 DNA template; 4 µL of Salmonella typhimurium DNA template; The above components were mixed by oscillation and then centrifuged, placed in a constant temperature metal bath, and amplified at 39°C for 20 minutes.
[0006] Furthermore, the bacterial liquid nucleic acid standard includes a bacterial liquid nucleic acid sample of Escherichia coli O157 and a bacterial liquid nucleic acid sample of Salmonella typhimurium.
[0007] Furthermore, the specific steps of the method for preparing the nucleic acid sample of Escherichia coli O157 bacterial liquid are as follows: (1) Use a sterile inoculation loop to pick 10-15 single clones from the plate, add them to 10 mL of liquid LB medium, and activate at 37°C until OD600 ≈ 0.6. This is used as the stock solution. (2) Dilute the bacterial solution 10-fold by adding 0.5 mL of bacterial solution to 4.5 mL of liquid LB medium. Select 100 µL of each of the three consecutive concentration gradients of diluted bacterial solution and inoculate them into the culture dish to ensure uniform distribution. (3) After overnight incubation at 37°C, count the number of colonies and calculate the bacterial content in the stock solution sample; (4) Take 100 µL of each diluted bacterial solution and place it in a centrifuge tube. Heat it in a water bath at 95°C for 10 minutes for thermal lysis. This will serve as the bacterial solution nucleic acid sample of Escherichia coli O157.
[0008] Furthermore, the specific steps of the method for preparing a nucleic acid sample of a bacterial liquid of Salmonella typhimurium are as follows: (1) Use a sterilized inoculation loop to pick 10-15 single clones from the plate, add them to 10 mL of liquid LB medium, and activate at 37°C until OD600≈0.6. (2) Dilute the bacterial solution 10-fold in a gradient of 10:1:500 μL of bacterial solution plus 4.5 mL of liquid LB medium. Select 100 μL of each of the three consecutive concentration gradient dilutions and inoculate them onto SS agar plates to ensure uniform distribution. (3) After overnight incubation at 37°C, count the number of colonies and calculate the bacterial content in the sample; (4) The quantified bacterial solution was heated at 95°C for 10 min to lyse the solution and use it as the nucleic acid sample of the Salmonella typhimurium bacterial solution.
[0009] The above technical solution can achieve the following beneficial effects: This study established a single-tube fluorescence colorimetric assay based on RPA-Exo technology for the early detection of foodborne pathogens Escherichia coli O157 and Salmonella typhimurium. This method enables multiple pathogen detection in a single tube using color-coded readouts by the naked eye. Furthermore, because the RPA enzyme and Exonuclease III exonuclease have the same reaction temperature of 37°C, they offer excellent single-tube compatibility. This eliminates the need for liquid transfer or column purification, significantly reducing detection time.
[0010] At the same time, the diagnostic method established by the present invention has a sensitivity of 10 for plasmids. 0 The sensitivity of nucleic acid in bacterial suspension is 10 copies / µL. 2 CFU / mL can ensure effective positive detection when the abundance of Escherichia coli O157 and Salmonella Typhimurium in the sample is low. The amplification test can be completed in only 20 minutes, which is much shorter than the 1 hour of conventional fluorescent PCR. At the same time, the reagents can be transported at room temperature and can maintain good performance and stability even under high temperature conditions of 37°C. This method has low requirements for temperature control of instruments and equipment, does not require the setting of complex procedures, and is very easy to operate. Therefore, this study established a rapid detection method for Escherichia coli O157 and Salmonella Typhimurium based on RPA-Exo technology with good performance. It can be applied to a variety of scenarios such as food safety emergencies and daily food safety testing, and plays a certain role in the detection and monitoring of Escherichia coli O157 and Salmonella Typhimurium. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 Schematic diagram of the visualized fluorescence dual RPA-Exo method.
[0012] Figure 2 Schematic diagram of dual detection of fluorescence signals for foodborne pathogens.
[0013] Figure 3 Visualizing the sensitivity of fluorescent Duplex RPA-Exo.
[0014] Figure 4 Fluorescence signal diagrams for specific verification of a series of foodborne pathogens. DETAILED DESCRIPTION
[0015] 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.
[0016] 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.
[0017] 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.
[0018] As attached Figure 1-4 As shown, a combined detection method for Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence is designed. Two high-discrimination molecular beacons are designed based on optical principles, and two cleverly designed probes are used for precise specific cutting to achieve the purpose of detecting Escherichia coli O157 and / or Salmonella typhimurium in one reaction. The probes include O157-Exo probe, rfb The E-Exo probe and its corresponding probe complementary long primers are O157-Exo probe complementary primer, rfb The complementary primers of E-Exo probes are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively. The primers include O157-F, O157-R, rfb EF, rfb ER, whose gene sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 respectively; the sequences are shown in Table 1-2 below:
[0019] Table 1 Probe and probe-complementary long primer sequences
[0020] Table 2 Primer sequences As a preferred embodiment, the detection method comprises the following steps: Preparation of double-stranded Exo fluorescent probe standards; Preparation of plasmid standards; Preparation of bacterial liquid nucleic acid standards; Application of dual fluorescence RPA-Exo reaction system; Fluorescence was generated by irradiation with blue light and a fluorescence signal graph was produced.
[0021] As a preferred embodiment, the application of the dual fluorescence RPA-Exo reaction system includes the detection of Escherichia coli O157 and / or Salmonella typhimurium. In 50 μL, the components are shaken and mixed, centrifuged, placed in a constant temperature metal bath, and amplified at 39°C for 20 minutes, wherein each component includes 10 μM primers O157-F, O157-R, rfb EF, rfb ER 1µL each; 10µM O157-Exo probe 0.3µL, rfb E-Exo probe 0.3µL; A buffer 25µL; ddH2O 9.9µL; B buffer 2.5µL; Escherichia coli O157 DNA template 4µL; Salmonella typhimurium DNA template 4µL.
[0022] As a preferred embodiment, the bacterial liquid nucleic acid standard includes a bacterial liquid nucleic acid sample of Escherichia coli O157 and a bacterial liquid nucleic acid sample of Salmonella typhimurium.
[0023] As a preferred embodiment, the method for preparing a nucleic acid sample of Escherichia coli O157 bacterial liquid comprises the following specific steps: (1) Use a sterile inoculation loop to pick 10-15 single clones from the plate, add them to 10 mL of liquid LB medium, and activate at 37°C until OD600 ≈ 0.6. This is used as the stock solution. (2) Dilute the bacterial solution 10-fold by adding 0.5 mL of bacterial solution to 4.5 mL of liquid LB medium. Select 100 µL of each of the three consecutive concentration gradients of diluted bacterial solution and inoculate them into the culture dish to ensure uniform distribution. (3) After overnight incubation at 37°C, count the number of colonies and calculate the bacterial content in the stock solution sample; 100 µL of each diluted bacterial solution was placed in a centrifuge tube, and heated in a water bath at 95°C for 10 min for thermal lysis to obtain a bacterial solution nucleic acid sample of Escherichia coli O157.
[0024] As a preferred embodiment, the method for preparing a nucleic acid sample of a Salmonella typhimurium bacterial liquid comprises the following specific steps: (1) Use a sterilized inoculation loop to pick 10-15 single clones from the plate, add them to 10 mL of liquid LB medium, and activate at 37°C until OD600≈0.6. (2) Dilute the bacterial solution 10-fold in a gradient of 10:1:500 μL of bacterial solution plus 4.5 mL of liquid LB medium. Select 100 μL of each of the three consecutive concentration gradient dilutions and inoculate them onto SS agar plates to ensure uniform distribution. (3) After overnight incubation at 37°C, count the number of colonies and calculate the bacterial content in the sample; (4) The quantified bacterial solution was heated at 95°C for 10 minutes to lyse the solution and obtain a nucleic acid sample of Salmonella typhimurium. 1. Reagents and Instruments RAA fluorescence amplification reagent was purchased from Hangzhou Zhongce Biotechnology Co., Ltd.; Exonuclease III exonuclease was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; Annealing Buffer for DNA Oligos (5X) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; SS agar medium was purchased from Changde Beekman Biotechnology Co., Ltd.; Top 10 cloning competent cells were purchased from Thermo Fisher Scientific.
[0025] 2. Preparation of double-stranded Exo fluorescent probe standards Anhui General Biotechnology Co., Ltd. synthesized long primers that complement the base pairing of the single-stranded Exo fluorescent probe. Double-stranded Exo probes were synthesized by high-temperature annealing. The long primers and Exo probes were diluted to 50 µM using sterile Milli-Q or distilled water. Annealing Buffer for DNA Oligos (5X) (Shanghai Biotech Co., Ltd.) was dissolved and mixed thoroughly for later use.
[0026] The annealing reaction system consists of 40 µL of sterile water, 20 µL of 5× annealing buffer, 20 µL of 50 µM Exo probe, and 20 µL of 50 µM Exo probe complementary primer, for a total of 100 µL. Add the reagents in the order listed above and mix thoroughly. Follow the annealing PCR protocol: fully denature the oligo at 95°C for 2 minutes, then decrease the temperature by 1°C every 90 seconds until it reaches 25°C. Anneal for approximately 90 minutes. Purify the annealed product using a DNA purification kit and use it as a double-stranded Exo fluorescent probe standard.
[0027] 3. Preparation of Plasmid Standards In order to establish a dual detection system for Escherichia coli O157 and Salmonella typhimurium and evaluate the specificity, sensitivity and quantitative accuracy of the present invention, a double detection system containing Escherichia coli O157 was prepared. rfbE gene standard plasmid and Salmonella typhimurium containing STM4497 gene standard plasmid.
[0028] choose rfb The 1095 bp gene fragment of the E gene sequence was identified as specific after Blast alignment. Its gene sequence is shown in SEQ ID NO.9, see Table 3 below. This gene fragment was constructed into the pUC19 cloning vector as a plasmid standard. rfb E standard plasmid (~5µg) was added to 50µL ddH2O and diluted to a concentration of 100ng / µL, which was used as the reference plasmid. The constructed E. coli O157 recombinant positive reference plasmid was serially diluted, and pUC19- rfb The full length of the E standard plasmid sequence is 3781 bp, according to the formula: (6.02×10 23 copies / mol) × plasmid concentration (ng / µL) × 10 -9 / (DNA length × 660) = copies / µL. Calculate the copy number concentration (copies / µL) corresponding to different recombinant positive reference plasmid mass concentrations.
[0029] The STM4497 gene (610 bp) was selected as the specific target fragment. Its gene sequence is shown in SEQ ID NO. 10 (see Table 3 below). This fragment was constructed into the pUC19 cloning vector and used as a standard. The pUC19-STM4497 standard plasmid (~5 µg) synthesized by General Bio was added to 50 µL of ddH2O and diluted to a concentration of 100 ng / µL to serve as the reference plasmid. The constructed Salmonella typhimurium recombinant positive reference plasmid was serially diluted. The pUC19-STM4497 standard plasmid sequence is 3296 bp long and is expressed according to the formula: (6.02 × 10 23 copies / mol) × plasmid concentration (ng / µL) × 10 -9 / (DNA length × 660) = copies / µL. Calculate the copy number concentration (copies / µL) corresponding to different recombinant positive reference plasmid mass concentrations.
[0030]
[0031] Table 3 rfb E gene (1095 bp) and STM4497 gene (610 bp) sequences IV. Preparation of Bacterial Liquid Nucleic Acid Standards Nucleic acid standards of Escherichia coli O157 and Salmonella typhimurium were prepared respectively to simulate the complexity of real samples and adapt to the RPA-Exo detection method. The viable bacterial concentration (CFU / mL) was determined by plate counting and compared with the copy number (copies / μL) of nucleic acid detection to verify the linear relationship of the quantitative method and achieve standardization and quantitative accuracy.
[0032] Escherichia coli O157 was used as a reference biological standard in this study. After inoculation onto LB solid medium plates and overnight culture at 37°C, plate counts were performed. Specifically, 10–15 single colonies were picked from the plate using a sterile inoculating loop and added to 10 mL of liquid LB medium. The culture was activated at 37°C until the OD600 reached approximately 0.6, which served as the stock solution. The culture solution was then serially diluted 10-fold, with 0.5 mL of the culture solution added to 4.5 mL of liquid LB medium. 100 µL of each of three consecutive dilutions was inoculated onto a Petri dish, ensuring even distribution. After overnight culture at 37°C, the number of colonies was counted to calculate the bacterial count in the stock solution. 100 µL of each diluted solution was placed in a centrifuge tube and heated in a water bath at 95°C for 10 minutes for thermal lysis. This served as the E. coli O157 nucleic acid sample.
[0033] Salmonella typhimurium was used as the reference biological standard for this study. After overnight culture at 37°C on SS agar plates, plate counts were performed. Specifically, 10–15 single colonies were picked from the plate using a sterile inoculating loop and added to 10 mL of liquid LB medium. The culture was activated at 37°C until the OD600 was approximately 0.6. The culture was then diluted 10-fold in a 500 µL to 4.5 mL LB medium dilution series. 100 µL of each of three consecutive dilutions was inoculated onto SS agar plates, ensuring even distribution. After overnight culture at 37°C, the number of colonies was counted to calculate the bacterial count in the sample. The quantified culture was then lysed by heating at 95°C for 10 minutes to serve as the Salmonella typhimurium nucleic acid sample. Example
[0034] Example 1: Feasibility verification of multiple fluorescence strategy: The key to the color-coding strategy used in this invention is the selection of an appropriate fluorescent signal to distinguish the presence of either or both targets in a single test tube. From available fluorophores, FAM (wavelength = 520 nm, green) and Texas Red (wavelength = 61 nm, red) were chosen to indicate Escherichia coli O157 and Salmonella typhimurium, respectively, with the expectation of producing yellow fluorescence when both targets are present. To validate these choices, long primers were synthesized that complement the base pairing of the single-stranded Exo probe. Double-stranded Exo probes were then synthesized by high-temperature annealing.
[0035] Since Exonuclease III is a nuclease exonuclease that acts on double-stranded DNA and has no activity on single-stranded DNA, Exonuclease III will specifically cut the molecular beacon under the guidance of the THF residue of the double-stranded Exo probe, and the resulting color will be observed. As expected, when dsDNA-MB (FAM) is specifically cut by the corresponding guided Exonuclease III, dsDNA-MB (FAM) emits green fluorescence, while dsDNA-MB (Texas Red) emits red fluorescence, and the centrifuge tube with the two cut MBs shows a clearly distinguishable yellow fluorescence, as shown in Figure 2. Figure 1 shown.
[0036] Example 2: Dual fluorescence RPA-Exo reaction system for detection of Escherichia coli O157 and Salmonella typhimurium Dual fluorescence RPA-Exo reaction system (total volume 50µL): 10µM primers O157-F, O157-R, rfb EF, rfb 1µL each of ER, 0.3µL of 10µM O157-Exo probe, rfb 0.3µL of E-Exo probe, 25µL of A Buffer, 9.9µL of ddH2O, 2.5µL of B Buffer, and 4µL each of Escherichia coli O157 and Salmonella typhimurium DNA templates. The above components were mixed by oscillation using a shaker and centrifuged, and then placed in a constant temperature metal bath for amplification. The amplification conditions were: 39°C for 20min. ddH2O was used instead of template DNA as a negative control. The reaction results were evaluated by the fluorescence generated by blue light irradiation. The sample that had reacted for 20min was taken out and placed under a blue light gel cutting instrument and irradiated with blue light, and pictures were taken with a smartphone under dark field conditions. Data processing and drawing were performed according to the following method: Image J software was used to uniformly adjust the grayscale of the image. The fluorescence value of each sample was read numerically using the lasso tool. Then the fluorescence signals under the same template DNA concentration conditions in the parallel repeated experiments were compared, and the average value was taken as the final experimental data under the template DNA concentration. After processing, each set of experimental data was made into an intuitive end-point fluorescence signal bar graph, as shown in the following figure. Figure 2 As shown in Figure 2, the fluorescence endpoint results showed that the two Exo probes specifically bound to the target sequences of Escherichia coli O157 and Salmonella typhimurium, respectively, and were recognized and cleaved by Exonuclease III, generating corresponding fluorescence signals. Figure 2 AB; and the yellow fluorescence produced by double target amplification and enzyme digestion has significant differentiation, see Figure 2C; It shows that the established dual RPA-Exo method is feasible for simultaneous typing and detection of Enterobacter O157 and Salmonella Typhimurium.
[0037] Example 3: Sensitivity of the RPA-Exo method for dual pathogen detection The concentration used is 10 0 -10 4 The sensitivity of the method was evaluated by averaging 100 copies / µL of standard plasmid DNA samples. Figure 3 As shown, where: (AB) RPA-Exo sensitivity to a single target. Detection 10 0 -10 4 Fluorescence endpoint histograms of RPA-Exo results for plasmid DNA standards cloned from E. coli O157 (A) or Salmonella typhimurium (B). (C) RPA-Exo sensitivity for dual targets. 0 -10 4 Endpoint signal (presented in histogram) and image of a 1:1 mixed plasmid DNA standard of E. coli O157 and Salmonella typhimurium. (DE) RPA-Exo sensitivity to a single target. Detection of 10 1 -10 4 Fluorescence endpoint bar graph and image of RPA-Exo results for CFU Escherichia coli O157 (A) or Salmonella typhimurium (B) bacterial suspension DNA standards. (F) RPA-Exo sensitivity for dual targets. RPA-Exo detection 10 1 -10 4 Endpoint signals (presented in histograms) and images of CFU E. coli O157 and Salmonella Typhimurium bacterial suspension DNA standards.
[0038] Fluorescence histograms and endpoint images indicate that the sensitivity for E. coli O157 as a single target reaches a theoretical maximum of 10 0 Copy / React ( Figure 3 A) and the sensitivity of Salmonella typhimurium as a single target also reached the theoretical maximum of 10 0 Copy / React ( Figure 3 B). When both targets are present, the sensitivity remains stable, also reaching 10 0 Copy / React ( Figure 3 C).
[0039] The concentration used is 10 0 -10 4 The sensitivity of the method was further evaluated by using CFU / mL bacterial solution nucleic acid standards. The results showed that the sensitivity of the method was 10 for both single target Escherichia coli O157 and Salmonella typhimurium. 2CFU / mL. Sensitivity remained unchanged for dual targets ( Figure 3 DF).
[0040] Example 4: Specificity of the RPA-Exo method for dual pathogen detection The specificity of the RPA-Exo method was verified by a series of common foodborne pathogens and zoonotic pathogens. Staphylococcus aureus ( S. aureus ), Listeria monocytogenes ( L. monocytogenes ), Escherichia coli ( E. coli ) and Brucella ( Brucella Common zoonotic pathogens include Aeromonas hydrophila ( A. hydrophila ), Edwardsiella tarda ( E. tarda )、Vibrio vulnificus( V. vulnificus ), Vibrio parahaemolyticus ( V. parahaemolyticus ) and Vibrio cholerae ( V. cholerae ).like Figure 4 As shown, the results showed that no fluorescent signals were generated when used to detect other pathogens, indicating that the method has good specificity.
[0041] 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 combined detection method for Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence, characterized by: The detection method designs two high-discrimination molecular beacons based on optical principles, and uses two cleverly designed probes to perform precise specific cutting to achieve the purpose of detecting Escherichia coli O157 and / or Salmonella typhimurium in one reaction. The probes include O157-Exo probe, rfb The E-Exo probe and its corresponding probe complementary long primers are O157-Exo probe complementary primer, rfb E-Exo probe complementary primers, primers include O157-F, O157-R, rfb EF, rfb ER.
2. The combined detection method for Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence according to claim 1, characterized in that: The O157-Exo probe, rfb E-Exo probe, O157-Exo probe complementary primers and rfb The gene sequences of the complementary primers of the E-Exo probe are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively; the O157-F, O157-R, rfb EF, rfb The ER gene sequences are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.
3. The combined detection method of Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence according to claim 1 or 2, characterized in that: The steps include: (1) Preparation of double-stranded Exo fluorescent probe standards; (2) Preparation of plasmid standards; (3) Preparation of bacterial liquid nucleic acid standards; (4) Application of dual fluorescence RPA-Exo reaction system; (5) Generate fluorescence by blue light irradiation and produce a fluorescence signal graph.
4. The combined detection method of Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence according to claim 3, characterized in that: The application of the dual fluorescence RPA-Exo reaction system, including the detection of Escherichia coli O157 and / or Salmonella typhimurium, is based on 50 μL. The specific steps are as follows: 10µM primers O157-F, O157-R, rfb EF, rfb 1 µL each for ER; 10µM O157-Exo probe 0.3µL, rfb E-Exo probe 0.3µL; A Buffer 25µL; ddH2O 9.9µL; B Buffer 2.5µL; 4 µL of Escherichia coli O157 DNA template; 4 µL of Salmonella typhimurium DNA template; The above components were mixed by oscillation and then centrifuged, placed in a constant temperature metal bath, and amplified at 39°C for 20 minutes.
5. The combined detection method of Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence according to claim 3, characterized in that: The bacterial liquid nucleic acid standard includes a bacterial liquid nucleic acid sample of Escherichia coli O157 and a bacterial liquid nucleic acid sample of Salmonella typhimurium.
6. The combined detection method of Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence according to claim 5, characterized in that: The specific steps of the method for preparing the nucleic acid sample of Escherichia coli O157 bacterial liquid are as follows: (1) Use a sterile inoculation loop to pick 10-15 single clones from the plate, add them to 10 mL of liquid LB medium, and activate at 37°C until OD600 ≈ 0.
6. This is used as the stock solution. (2) Dilute the bacterial solution 10-fold by adding 0.5 mL of bacterial solution to 4.5 mL of liquid LB medium. Select 100 µL of each of the three consecutive concentration gradients of diluted bacterial solution and inoculate them into the culture dish to ensure uniform distribution. (3) After overnight incubation at 37°C, count the number of colonies and calculate the bacterial content in the stock solution sample; (4) Take 100 µL of each diluted bacterial solution and place it in a centrifuge tube. Heat it in a water bath at 95°C for 10 minutes for thermal lysis to obtain the bacterial solution nucleic acid sample of Escherichia coli O157.
7. The combined detection method of Escherichia coli O157 and Salmonella typhimurium based on RPA-Exo multiple fluorescence according to claim 5, characterized in that: The specific steps of the method for preparing a nucleic acid sample of a bacterial liquid of Salmonella typhimurium are as follows: (1) Use a sterilized inoculation loop to pick 10-15 single clones from the plate, add them to 10 mL of liquid LB medium, and activate at 37°C until OD600≈0.
6. (2) Dilute the bacterial solution 10-fold in a gradient of 10:1:500 μL of bacterial solution plus 4.5 mL of liquid LB medium. Select 100 μL of each of the three consecutive concentration gradient dilutions and inoculate them onto SS agar plates to ensure uniform distribution. (3) After overnight incubation at 37°C, count the number of colonies and calculate the bacterial content in the sample; (4) The quantified bacterial solution was heated at 95°C for 10 min to lyse the solution and obtain a nucleic acid sample of the Salmonella typhimurium bacterial solution.