A combined probe for detecting Aeromonas and its application

Abstract

The present invention discloses a combination probe for detecting Aeromonas bacteria and its application. The Aeromonas bacteria detection combination probe of the present invention comprises an analytical probe, a capture probe, and a signal probe. The analytical probe specifically binds to the rRNA of Aeromonas bacteria; the capture probe is connected to a carrier and specifically binds to one end of the analytical probe; and the signal probe is provided with a signal protein marker and specifically binds to the other end of the analytical probe. The Aeromonas detection combination probe of the present invention is cleverly designed and can rapidly detect Aeromonas bacteria qualitatively and quantitatively. The detection method is simple to operate, has good reproducibility, and is suitable for large-scale promotion and application.

Description

Technical Field

[0001] The present invention relates to a combined probe for detecting Aeromonas and application thereof, belonging to the technical field of molecular diagnosis. Background Art

[0002] The Chinese mitten crab, commonly known as river crab, is one of the most important crustaceans in my country's freshwater aquaculture. Aeromonas is a common opportunistic pathogen in water bodies. It has strong adaptability to the environment and is widely present in freshwater lakes, estuaries and other aquatic environments. When water quality deteriorates, Aeromonas can become the dominant flora in the water body, infecting aquatic animals, causing inflammation, sepsis, etc., affecting their immune system. In severe cases, large-scale deaths can occur, causing serious economic losses to the aquaculture industry. Existing detection technology involves strain cultivation of Aeromonas, followed by a series of tests such as biochemical identification. The operation is complicated, time-consuming, and very inconvenient.

[0003] Therefore, in order to effectively detect Aeromonas, there is an urgent need in this field to develop a simple and convenient Aeromonas detection method with good specificity and high sensitivity. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an Aeromonas bacteria detection combination probe, which can quickly detect Aeromonas bacteria qualitatively and quantitatively. The detection method is simple to operate and has good repeatability, and is suitable for large-scale promotion and application.

[0005] The first object of the present invention is to provide a combined probe for detecting Aeromonas bacteria, comprising an analytical probe, a capture probe and a signal probe, wherein the analytical probe specifically binds to the rRNA of Aeromonas bacteria; the capture probe is connected to a carrier and specifically binds to one end of the analytical probe; and the signal probe is provided with a signal protein marker and specifically binds to the other end of the analytical probe.

[0006] Furthermore, the nucleotide sequence of the analysis probe is shown in SEQ ID NO: 1.

[0007] Furthermore, the nucleotide sequence of the capture probe is shown in SEQ ID NO: 2.

[0008] Furthermore, the nucleotide sequence of the signal probe is shown in SEQ ID NO: 3.

[0009] Furthermore, the signal protein marker is a fluorescein marker.

[0010] Furthermore, the capture probe is connected to the carrier through biotin-streptavidin interaction.

[0011] A second object of the present invention is to provide a method for detecting Aeromonas bacteria using the Aeromonas bacteria detection combination probe, comprising the following steps:

[0012] S1: Take a certain amount of Aeromonas bacteria, lyse and disrupt the cell wall, and add lysis buffer containing S1 nuclease protection assay probe for gradient dilution to obtain cell wall disruption solution;

[0013] S2, incubating the analytical probe with the cell wall breaking solution, adding S1 nuclease for digestion after incubation, and denaturing the digestion product to obtain a denatured product;

[0014] S3, adding the denatured product to a carrier connected to a capture probe for hybridization, and washing the carrier after hybridization;

[0015] S4, adding a signal probe to the carrier washed in step S3 for hybridization, and washing the carrier after hybridization;

[0016] S5, adding an enzyme-labeled antibody against the signal protein marker to the carrier washed in step S4, hybridizing with the signal protein marker, and washing the carrier after hybridization;

[0017] S6, adding the enzyme-labeled substrate to the carrier after washing in step S5 to perform a color reaction, detecting the absorbance after the reaction, and preparing a standard curve;

[0018] S7. Lyse and disrupt the cell wall of the sample to be tested, and add a lysis buffer containing an S1 nuclease protection analysis probe to obtain a disrupted cell wall solution to be tested. Detect the absorbance value according to steps S2 to S6, and determine the amount of Aeromonas bacteria in the sample to be tested by comparing with the standard curve.

[0019] Furthermore, the enzyme label is a horseradish peroxidase label, and the substrate of the enzyme label is tetramethylbenzidine or o-phenylenediamine.

[0020] Furthermore, in step S3, the hybridization conditions are 94-96°C for 14-16 min and 28-32°C in a water bath for 0.8-1.2 h.

[0021] Furthermore, in step S4, the hybridization conditions are 42-44°C for 0.8-1.2 h, and in step S5, the hybridization conditions are 42-44°C for 0.4-0.6 h.

[0022] The beneficial effects of the present invention are:

[0023] The Aeromonas detection combination probe of the present invention is cleverly designed and can quickly detect Aeromonas qualitatively and quantitatively. The detection signal is linearly related to the number of Aeromonas bacteria in aquatic products. 2=0.996, and the detection method is simple to operate and has good repeatability, making it suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the detection of various aquatic bacteria using a combination of probes for Aeromonas. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0026] In this embodiment, a specific probe was used to detect Aeromonas genus, a foodborne pathogen unique to aquatic products, especially Chinese mitten crab, to generate a standard curve and verify its specificity.

[0027] The specific steps for preparing the standard curve are:

[0028] The logarithmic phase Aeromonas hydrophila bacteria were processed to obtain their rRNA and diluted into gradient concentrations;

[0029] hybridizing the analytical probe with the rRNA to form a DNA / RNA hybrid;

[0030] adding nucleic acid S1 nuclease to the DNA / RNA hybrid product for digestion treatment, digesting the unmatched single-stranded DNA or RNA and the DNA / RNA hybrid to obtain DNA / RNA hybrids equal in number to the target RNA molecule;

[0031] Denaturing the digestion product DNA / RNA hybrid to obtain single-stranded DNA and single-stranded RNA. The denaturation method can adopt a denaturation technique commonly used in the field, such as a heat denaturation method;

[0032] hybridizing the denatured product with the capture probe immobilized on the carrier, and then washing; forming a DNA / DNA hybrid after hybridization;

[0033] Adding the signal probe and hybridizing with the analysis probe, followed by washing; forming a "sandwich" hybridization complex after hybridization;

[0034] adding an enzyme-labeled antibody against the signal protein marker to hybridize with the protein marker, and then washing;

[0035] The enzyme-labeled substrate is added to develop color, and the light absorbance at 450 nm and 630 nm is measured, and the rRNA concentration is determined based on the absorbance.

[0036] Example 1

[0037] 1. Design and synthesis of probes specific to the rRNA region of Aeromonas bacteria 16:

[0038] The rRNA sequences of 16 Aeromonas bacteria were compared with those of bacteria in other aquatic products. It was found that the rRNA sequences of 16 Aeromonas bacteria were complementary to all species of Aeromonas, but had no homology with nucleotide sequences of bacteria in other major aquatic products. It was determined to be a specific sequence, and the analysis probe was designed as follows:

[0039] SEQ ID NO: 1:

[0040] 5'-AGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGT-3' (see the nucleotide sequence shown in SEQ ID NO: 1);

[0041] 2. Based on the above analytical probes, design and synthesize other probes required for detection:

[0042] The capture probe is SEQ ID NO: 2, complementary to the 3' end of the analytical probe, and labeled with biotin at the 5' end;

[0043] 5'-ACCGTGTCTCAGTTCCAGTG-3' (see the nucleotide sequence shown in SEQ ID NO: 2);

[0044] The signal probe is SEQ ID NO: 3, complementary to the 5' end of the analytical probe, and labeled with FITC isothiocyanate at the 3' end;

[0045] 5'-CCAGCTAGGGATCGTCGCCT-3' (see the nucleotide sequence shown in SEQ ID NO: 3)

[0046] 3. Preparation stage:

[0047] Streptavidin was prepared with coating buffer (CBS) at 10 μg / mL, and 150 μL was added to each well of the microplate. The plate was sealed and stored at 4°C for 24 h. The plate was then washed three times with PBST (0.01 mol / L PBS buffer containing 0.5% Tween-20), blocked with 250 μL of 0.1% BSA at 37°C for 60 min, and finally washed three times with PBST to prepare an avidin-coated plate. The plate was dried, sealed, and stored at 4°C until use.

[0048] Prepare 1000 nmol / L capture probe solution, take 100 μL capture probe solution and add it to streptomycin-coated microplate, incubate at 37°C for 2 h, and rinse three times with PBST to immobilize the capture probe, then store at 4°C until use.

[0049] Count the logarithmic phase Aeromonas (Aeromonas hydrophila was used as the experimental bacterium) under a microscope and calculate the bacterial concentration. Centrifuge 5 mL of the bacterial solution (8000 g, 10 min). Remove the supernatant and add 2 mL of lysis buffer (80% formamide, 450 mM NaCl, 5 mM Na2EDTA, 1 mg / mL yeast tRNA, 1% SDS, pH 6.4) and disrupt the cells by ultrasonication. Collect the cell wall-broken solution for later use.

[0050] The Aeromonas samples collected by filtration were serially diluted using a lysis buffer containing an S1 nuclease protection assay probe.

[0051] Add 100 μL of Aeromonas cell wall disruption solution, 50 μL of mineral oil, and 15 μL of 1000 nmol / L analytical probe, and treat in a 95°C water bath for 15 min and then in a 30°C water bath for 1 h to allow the analytical probe to bind to the target sequence.

[0052] Add 100 μL of S1 nuclease and incubate in a 37°C water bath for 1 h to perform S1 nuclease digestion.

[0053] Add 500 μL of stop buffer (62.5 mM NaOH, 30 mM EDTA, 0.5 M PBS, pH 7.2) and incubate at 95°C for 15 minutes to denature the DNA-RNA hybrid into single-stranded DNA and RNA. Simultaneously, the single-stranded RNA is degraded, leaving only the analytical probe bound to the target sequence in the solution.

[0054] 4. Sandwich hybridization stage:

[0055] 100 μL of the above reaction mixture was transferred to the enzyme-labeled plate pre-immobilized with the capture probe, hybridized at 43°C for 1 hour, and then washed three times with PBS buffer to allow the capture probe to bind to the analysis probe.

[0056] Hybridization buffer (4 × SSC, 10% formamide, 0.02% SDS, pH 7.2) containing 100 μL of 1000 nM signal probe was added to each well, hybridized at 43°C for 30 min, and washed three times with PBS to form a sandwich hybridization structure.

[0057] 5. Signal detection phase:

[0058] 100 μL of anti-fluorescein antibody labeled with horseradish peroxidase (1:6000 diluted in PBS, 0.1% bovine serum albumin) was added to each well and incubated at 37°C for 30 min. The wells were then washed four times with PBS to allow the anti-fluorescein antibody to bind to the signal probe.

[0059] Add 100 μL of TMB colorimetric solution to each well (prepare TMB to a 1% concentration using anhydrous ethanol. Before use, add 0.1 mL of 1% TMB solution to 9.9 mL of pH 5.0 phosphate-citrate buffer, then add 1 μL of 30% H2O2 per mL of TMB, mix well, and use immediately). After treating at 37°C for 15 min, add 100 μL of 2 mol / L sulfuric acid to each well to terminate the reaction, and measure the absorbance at 450 nm and 630 nm.

[0060] 6. Results

[0061] Based on the probe design principles, we designed analytical probes for Aeromonas (Table 1). The main probe is complementary to the conserved fragment of Aeromonas 16S rRNA, the capture probe is complementary to the 3' end of the main probe and labeled with biotin at the 5' end, and the signal probe is complementary to the 5' end of the main probe and labeled with anti-fluorescein antibody (FITC) at the 3' end. The hybridization temperature when the probe hybridizes with the corresponding rRNA or probe DNA is calculated based on the ionic strength and formamide content set in this experiment.

[0062] Table 1 Detection probes for Aeromonas

[0063]

[0064] Test results such as Figure 1 shown.

[0065] Figure 1 The results of detecting various aquatic bacteria using the detection combination probe for Aeromonas are shown, demonstrating that the method of the present invention has extremely high feasibility and specificity.

[0066] Therefore, the Aeromonas detection combination probe of the present invention can be used for qualitative and quantitative detection of Aeromonas. First, the 16S rRNA target sequence is extracted, the analysis probe is hybridized, the nucleic acid S1 nuclease removes mismatches and single-stranded DNA, and after denaturation, it is hybridized with the capture probe, and finally hybridized with the signal probe containing a fluorescent substance. Through the specificity of the protein macromolecule and the antibody, a detection signal is finally obtained in the color development system, and the type and quantity of rRNA can be determined.

[0067] In summary, the Aeromonas detection combination probe of the present invention is cleverly designed and can rapidly detect Aeromonas qualitatively and quantitatively. The detection method is simple to operate and has good repeatability, making it suitable for large-scale promotion and application.

[0068] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A combined probe for detecting Aeromonas bacteria, characterized in that: It comprises an analysis probe, a capture probe and a signal probe, wherein the analysis probe specifically binds to the rRNA of Aeromonas bacteria; the capture probe is connected to a carrier and specifically binds to one end of the analysis probe; the signal probe is provided with a signal protein marker and specifically binds to the other end of the analysis probe, the nucleotide sequence of the analysis probe is shown in SEQ ID NO: 1, the nucleotide sequence of the capture probe is shown in SEQ ID NO: 2, and the nucleotide sequence of the signal probe is shown in SEQ ID NO:

3.

2. The combined probe for detecting Aeromonas bacteria according to claim 1, characterized in that: The signal protein marker is a fluorescent marker.

3. The combined probe for detecting Aeromonas bacteria according to claim 1, characterized in that: The capture probe is connected to the carrier through biotin-streptavidin interaction.

4. A method for detecting Aeromonas bacteria using the Aeromonas bacteria detection combination probe according to any one of claims 1 to 3, characterized in that: The steps include: S1: Take a certain amount of Aeromonas bacteria, lyse and disrupt the cell wall, and add lysis buffer containing S1 nuclease protection assay probe for gradient dilution to obtain cell wall disruption solution; S2, incubating the analytical probe with the cell wall breaking solution, adding S1 nuclease for digestion after incubation, and denaturing the digestion product to obtain a denatured product; S3, adding the denatured product to a carrier connected to a capture probe for hybridization, and washing the carrier after hybridization; S4, adding a signal probe to the carrier washed in step S3 for hybridization, and washing the carrier after hybridization; S5, adding an enzyme-labeled antibody against the signal protein marker to the carrier washed in step S4, hybridizing with the signal protein marker, and washing the carrier after hybridization; S6, adding the enzyme-labeled substrate to the carrier after washing in step S5 to perform a color reaction, detecting the absorbance after the reaction, and preparing a standard curve; S7. Lyse and disrupt the cell wall of the sample to be tested, and add a lysis buffer containing an S1 nuclease protection analysis probe to obtain a disrupted cell wall solution to be tested. Detect the absorbance value according to steps S2 to S6, and determine the amount of Aeromonas bacteria in the sample to be tested by comparing with the standard curve.

5. The method according to claim 4, characterized in that The enzyme label is a horseradish peroxidase label, and the substrate of the enzyme label is tetramethylbenzidine or o-phenylenediamine.

6. The method according to claim 4, characterized in that In step S3, the hybridization conditions are 94-96°C for 14-16 min and 28-32°C in a water bath for 0.8-1.2 h.

7. The method according to claim 4, characterized in that In step S4, the hybridization conditions are 42-44°C for 0.8-1.2 h, and in step S5, the hybridization conditions are 42-44°C for 0.4-0.6 h.

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