Kit for detecting shigella by Marker method and detection method

Abstract

The invention provides a kit for detecting shigella by a Marker method, and the Marker method represents fluorescence intensity corresponding to shigella with different concentrations and is a basis for establishing a Marker graph; the kit comprises a Marker graph, a magnetic aptamer, a detection aptamer, a bacterial lysis solution, an enzyme digestion solution, a binding solution, a washing solution and a magnetic frame. The detection method comprises the following steps: the kit is used for splitting the shigella through a bacterial lysis solution to release DNA, the DNA is cut through an enzyme digestion solution to obtain a fragment rich in an ipaH gene, the magnetic aptamer and the detection aptamer are specifically combined with the ipaH fragment under the action of a binding solution to form a compound, the compound is washed with washing liquid, the fluorescence intensity is detected and compared with a Marker diagram, and quantitative detection of the shigella is realized. The method does not need to treat a sample in advance, shortens the detection time, and has strong specificity and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of foodborne pathogenic bacteria analysis and detection, and particularly relates to a kit and a detection method for detecting Shigella using a marker method. Background Art

[0002] Shigella ( Shigella spp Shigella spp. is a highly contagious enteric pathogen that can cause severe intestinal illnesses, including bacillary dysentery and acute diarrhea, characterized by fever, abdominal pain, and diarrhea. The National Food Safety Standard GB4789.5-2012 specifies the testing method for Shigella in foods. The detection limit for Shigella is set to detect extremely low numbers of bacteria to ensure food safety. Testing for Shigella has become a mandatory health indicator for food quality and safety monitoring.

[0003] The detection methods for Shigella include traditional culture methods, immunological methods and molecular biology methods. Traditional culture methods are cumbersome, time-consuming, and inaccurate, and cannot meet the needs of rapid and accurate testing. Although immunological and molecular biological methods have improved the sensitivity and specificity of detection, they are expensive and cannot quickly and accurately detect low concentrations of Shigella. Therefore, rapid, sensitive, and efficient detection methods are crucial to ensuring food quality and safety. ipaH The gene is a specific gene on its invasion plasmid and is widely used for Shigella-specific detection. Summary of the Invention

[0004] The present invention provides a kit and a method for detecting Shigella by a marker method, which can effectively detect Shigella in food with a detection limit of 10 1 cfu / mL.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A kit for detecting Shigella by a marker method, wherein the bacteria to be detected are lysed to release DNA, and enzyme digestion is performed to obtain a DNA-rich ipaH The gene fragments then combine with the magnetic aptamer and the detection aptamer to form a specific complex: magnetic aptamer- ipaH - Detect the aptamer, detect the fluorescence intensity after washing and compare it with the marker image to obtain the concentration of the bacteria to be detected.

[0006] The reagent kit for detecting Shigella by the marker method comprises: a marker map, a magnetic aptamer, a detection aptamer, a bacterial lysis solution, an enzyme digestion solution, a binding solution, a washing solution, and a magnetic stand.

[0007] The marker graph is based on the Shigella concentration of 10 1, 10 2 , 10 3 , 10 4 The fluorescence intensity value corresponding to cfu / mL was constructed.

[0008] The magnetic aptamer is based on ipaH The genetically designed oligonucleotide is modified with Biotin at the 5' end and connected to streptavidin magnetic beads, namely SA magnetic beads, with a structure of SA magnetic beads-Biotin-poly(dT)-ssDNA and a sequence of 5'-TTTTTTTCGGCAGTGCGGAGGTCAT-3'.

[0009] The detection aptamer is ipaH The genetically designed oligonucleotide is labeled with fluorescein (Fluo) at the 5' end, has a structure of Fluo-poly (dT)-ssDNA, and a sequence of 5'-AAGTCAGAACTCTCCATTTTTTTT-3'.

[0010] The bacterial lysis buffer included 0.8% SDS, 25 mM EDTA, 30 mM sodium citrate (pH = 8.0), 75 mM NaCl, 0.5% Triton X-100, and 1% β-mercaptoethanol.

[0011] The binding solution includes 60% formamide, 250mM NaH2PO4, 200mM NaCl, 15mM sodium citrate solution, pH=7.0; 60% formamide serves as a heat stabilizer to stabilize the structure of DNA.

[0012] The enzyme digestion solution was SmaI, NcoI, AvaI, and BspQI, with a concentration of 5 U / μL.

[0013] The washing solution was phosphate buffered saline (PBS), pH = 7.4.

[0014] The steps for constructing the Marker graph are as follows: (1) Determine the absorbance OD of the gradient dilution of Shigella culture 600 .

[0015] (2) After gradient dilution, plate the sample and count the colony counts (CFUs). A linear relationship between absorbance and colony count was established to determine the true concentration of the bacterial solution.

[0016] (3) Preparation of Shigella culture solutions of different concentrations: 10 1 , 10 2 , 10 3 , 10 4 cfu / mL.

[0017] (4) Use the kit method to detect the fluorescence intensity of each concentration of bacterial solution, establish a standard curve corresponding to the fluorescence intensity and the number of colonies, and verify it by the dilution plate method to obtain a marker graph; where Y1 on the left side of the marker graph is the fluorescence intensity value, and Y2 is the bacterial concentration.

[0018] The preparation of the magnetic aptamer comprises the following steps: Take 50 μL of 20 mg / mL SA magnetic beads in a 1.5 mL centrifuge tube, add 450 μL of buffer I, vortex and shake to mix, then place on a magnetic stand, magnetically separate and discard the supernatant; the buffer I contains 2.5 mM Tris-HCl, 1.0 mM EDTA, 1.0 M NaCl, pH = 7.5.

[0019] In step Buffer II was added to the centrifuge tube and the SA magnetic beads were mixed to a final concentration of 2 mg / mL; the buffer II included 5.0 mM Tris-HCl, 2.0 mM EDTA, 2.0 M NaCl, pH = 7.5.

[0020] Add 50 μL of specific aptamer Biotin-poly (dT)-ssDNA and rotate at room temperature for 30-60 minutes.

[0021] After magnetic separation, the supernatant was discarded, and the mixture was washed 2-3 times with buffer solution. 100 μL of buffer solution I was added and mixed to obtain the magnetic aptamer.

[0022] The detection probes are labeled with fluorescein FITC, phycoerythrin PE, tetramethylrhodamine azide TAMRA-N, and tetramethylrhodamine isothiocyanate TRITC, with a concentration of 50 mM.

[0023] A method for detecting Shigella using a marker method comprises the following steps: (1) Take 10 mL of the sample to be tested and filter it through an ultrafiltration membrane into a 1.5 mL centrifuge tube. Add 200 μL of bacterial lysis buffer and lyse it at room temperature for 10 min. Add 10 μL of enzyme digestion buffer and react at 37°C for 5 min.

[0024] (2) Add 200 μL of binding solution and react at 65°C for 30-60 min. Add 50 μL of magnetic aptamer and 50 μL of detection aptamer and react at room temperature for 10 min. Perform magnetic separation on a magnetic rack and discard the supernatant.

[0025] (3) Add 500 μL of washing solution and wash 2-3 times, then disperse in 100 μL of ddH2O to obtain magnetic aptamer- ipaH-Detect the aptamer complex; compare the fluorescence intensity with the marker image to determine the Shigella concentration of the sample to be tested.

[0026] The significant advantages of the present invention are: (1) The present invention uses the provided Marker graph for detection, which has high sensitivity, low detection limit, and a detection linear range of 10 1 -10 4 cfu / mL. The marker pattern formed by the marker method can effectively detect foodborne Shigella; it has important promoting significance for the development of foodborne pathogen analysis and detection technology and microbial detection technology.

[0027] (2) The marker map provided by the present invention is based on the Shigella-specific gene ipaH The designed magnetic aptamer and detection aptamer have strong specificity and can ensure the accuracy and specificity of the detection results.

[0028] (3) The present invention provides a detection kit for Shigella detection using dual aptamers, ensuring the accuracy and reliability of the detection results through dual specificity. The magnetic aptamer is pretreated and can be directly used to detect unenriched samples, reducing sample loss during the experiment and avoiding the use of organic reagents during the detection process. It also effectively avoids the impact of proteins and salts in the sample on the experimental results, shortening the detection cycle in practical applications. Since the magnetic aptamer is used in the detection kit, it can be used for downstream research such as high-throughput extraction, detection of biomolecules or targeted positioning of organisms, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the present invention; Figure 2 This is a transmission electron microscopy (TEM) image of the streptavidin magnetic beads prepared in the present invention; Figure 3 is the UV absorbance-Shigella colony count standard curve of the present invention; Figure 4 This is a fluorescence intensity diagram of Shigella bacterial solution detected by concentration gradient of the present invention; Figure 5 This is the standard fluorescence intensity marker image of Shigella of the present invention; Figure 6 This is the fluorescence intensity diagram of bacterial solution A and B of the present invention; Figure 7 This is a plate drawing of bacterial solutions A and B of the present invention; Figure 8 This is the specific verification result of the present invention. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] The following examples are provided to further illustrate the present invention but are not intended to limit the present invention in any way.

[0032] Example 1, preparation of a magnetic aptamer for detecting Shigella, comprising the following steps: Take 50 μL of 20 mg / mL SA magnetic beads in a 1.5 mL centrifuge tube, add 450 μL of buffer I, vortex and mix, then place on a magnetic stand for magnetic separation and discard the supernatant; the buffer I contains 2.5 mM Tris-HCl, 1.0 mM EDTA, 1.0 M NaCl, pH = 7.5; In step Buffer II was added to the centrifuge tube and the SA magnetic beads were mixed to a final concentration of 2 mg / mL; the buffer II included 5.0 mM Tris-HCl, 2.0 mM EDTA, 2.0 M NaCl, pH = 7.5.

[0033] Add 50 μL of specific aptamer Biotin-poly(dT)-ssDNA and rotate at room temperature for 30-60 minutes; After magnetic separation, the supernatant was discarded, and the solution was washed 2-3 times with buffer I. 100 μL of buffer I was added and mixed to obtain the magnetic aptamer.

[0034] Figure 2 TEM image of SA magnetic beads.

[0035] Example 2, establishment of a standard curve for the correlation between absorbance and bacterial count of Shigella: (1) Shigella was cultured in liquid for 24 h. 1 mL of bacterial suspension was placed in a centrifuge tube and centrifuged at 10,000 r / min for 5 min. The supernatant was discarded and the precipitate was retained. 1 mL of sterile saline (0.9%) was added to the precipitate and mixed evenly. 500 μL, 400 μL, 300 μL, 200 μL, and 100 μL were pipetted into 5 test tubes in sequence. Then, appropriate amounts of saline (0.9%) were added to each tube to make the total amount reach 1 mL. The mixture was thoroughly mixed to obtain a gradient dilution bacterial suspension.

[0036] (2) The gradient diluted bacterial suspension was placed in cuvettes and the absorbance was measured at a wavelength of 600 nm. Each sample was measured three times, and physiological saline was used as a blank control.

[0037] (3) 50 μL of the gradient dilution bacterial suspension was aspirated for plate counting, with each concentration repeated three times. A blank plate was used as a control. The colony count was calculated after incubation at 37°C for 48 h.

[0038] (4) Use the absorbance of the bacterial solution as the ordinate and the colony count (cfu) as the abscissa to establish a standard curve for the absorbance-bacterial count correlation of Shigella.

[0039] Figure 3 The standard curve of the Shigella absorbance-bacterial count correlation was established, and its linear equation was y=1.3104x-0.3076, with a correlation coefficient R 2 =0.9952; From the data, we can see that there is a good linear relationship between the number of bacteria and absorbance.

[0040] Example 3, a method for detecting Shigella using a marker image: (1) Take 10 mL of the sample to be tested and filter it through an ultrafiltration membrane into a 1.5 mL centrifuge tube. Add 200 μL of bacterial lysis buffer and lyse it at room temperature for 10 min. Add 10 μL of enzyme digestion buffer and react at 37°C for 5 min. (2) Add 200 μL of binding solution and react at 65°C for 30-60 min. Add 50 μL of magnetic aptamer and 50 μL of detection aptamer and react at room temperature for 10 min. Separate the mixture with a magnetic stand and discard the supernatant. (3) Add 500 μL of washing solution and wash 2-3 times, then disperse in 100 μL of ddH2O to obtain magnetic aptamer- ipaH - Detect the aptamer complex. Compare the fluorescence intensity with the marker image to determine the Shigella concentration in the sample to be tested.

[0041] Example 4, a method for constructing a standard fluorescence intensity marker graph: The specific steps are as follows: (1) The Shigella concentration was obtained according to the standard curve of the correlation between absorbance and bacterial count in Example 2. 1 , 10 2 , 10 3 , 10 4 cfu / mL concentration gradient bacterial solution, each gradient was repeated 3 times; (2) Add 200 μL of bacterial lysis buffer to the concentration gradient bacterial solution and lyse at room temperature for 10 min, add 10 μL of enzyme digestion buffer and react at 37°C for 5 min; add 200 μL of binding buffer and react at 65°C for 30-60 min, add 50 μL of magnetic aptamer and 50 μL of detection aptamer and react at room temperature for 10 min, and magnetically separate on a magnetic stand and discard the supernatant; (3) Add 500 μL of washing solution and wash 2-3 times, disperse in 100 μL of ddH2O, and detect the fluorescence intensity of bacterial solution with different concentrations. 1 , 10 2 , 10 3 , 10 4 The Marker graph was established by comparing the fluorescence intensity of the bacterial solution with the actual concentration of the bacterial solution at a concentration gradient of cfu / mL.

[0042] Figure 4 Detect the fluorescence intensity of bacterial suspensions with different concentrations. Figure 5 This is the established standard fluorescence intensity marker graph, where Y1 on the left is the fluorescence intensity value and Y2 is the bacterial concentration.

[0043] Example 5, the actual operation of detecting Shigella using the Marker method Shigella detection kit is as follows: (1) The bacterial solution cultured for 24 h was diluted to obtain two bacterial solutions of unknown concentrations, A and B. In view of the national standard for the limit of Shigella, the concentration was controlled at 10 1 -10 4 cfu / mL; (2) Detect bacterial solutions A and B according to the detection method of Example 3, and compare the concentrations of bacterial solutions A and B with the standard fluorescence intensity marker graph. Figure 6 The fluorescence intensity graph of bacterial solution A and B is compared with the marker graph to show that the fluorescence intensity of bacterial solution A is 5.9×10 3 cfu / mL, B is 3.1×10 2 cfu / mL.

[0044] Example 6: The accuracy and feasibility of a marker image for detecting Shigella were verified as follows: (1) Bacterial suspensions of unknown concentrations A and B were diluted in a gradient manner while being tested according to the method of Example 5. 100 μL of the suspension was applied to a plate and cultured in a constant temperature incubator at 37°C for 48 h. Physiological saline was used as a blank control.

[0045] (2) After the colonies are clear, count the number of colonies and calculate the concentrations of bacterial solutions A and B using the multiple dilution relationship.

[0046] (3) Compare the calculated concentrations of bacterial solutions A and B with the data detected in Example 5 to verify the accuracy and feasibility of the detection method of the present invention. Figure 7 The result is the colony count of the plate, and the result A is 6.16×10 3 cfu / mL, B is 3.32×10 2 cfu / mL; it can be seen that the research method of the present invention is consistent with the results of plate colony counting and has good feasibility.

[0047] Example 7: Validation of the specificity of a marker image for Shigella detection This example is to verify the anti-interference and specificity of the present invention. The detection steps are the same as those in Example 3, except that the target Shigella in the detection system is replaced by Staphylococcus aureus and Salmonella, respectively, and the bacterial concentration is obtained by comparing the fluorescence intensity with the marker graph. The fluorescence detection results are shown in Figure 2. Figure 8 As shown in the figure, the calculated concentrations of Staphylococcus aureus and Salmonella in the test system are close to 0 cfu / mL, while their actual concentrations are 10 7 -10 8 cfu / mL; this indicates that the detection method and kit have good specificity and excellent anti-interference ability.

Claims

1. A kit for detecting Shigella by a marker method, characterized in that: The marker method is to lyse the bacteria to be tested to release DNA, and then obtain DNA-rich ipaH The gene fragments then combine with the magnetic aptamer and the detection aptamer to form a specific complex: magnetic aptamer- ipaH - Detect the aptamer, detect the fluorescence intensity after washing and compare it with the marker image to obtain the concentration of the bacteria to be detected; The kit for detecting Shigella by the marker method comprises: a marker image, a magnetic aptamer, a detection aptamer, a bacterial lysis solution, an enzyme digestion solution, a binding solution, a washing solution, and a magnetic stand; The marker graph is based on the Shigella concentration of 10 1 -10 4 The fluorescence intensity value corresponding to cfu / mL was constructed; The magnetic aptamer is based on ipaH The genetically designed oligonucleotides were modified with Biotin at the 5' end and linked to streptavidin magnetic beads, namely SA magnetic beads, with the structure of SA magnetic beads-Biotin-poly(dT)-ssDNA and the sequence 5'-TTTTTTTCGGCAGTGCGGAGGTCAT-3'; The detection aptamer is ipaH The genetically designed oligonucleotides were labeled with fluorescein (Fluo) at the 5' end, with the structure of Fluo-poly(dT)-ssDNA and the sequence 5'-AAGTCAGAACTCTCCATTTTTTTT-3'; Bacterial lysis buffer included 0.8% SDS, 25 mM EDTA, 30 mM sodium citrate (pH = 8.0), 75 mM NaCl, 0.5% Triton X-100, and 1% β-mercaptoethanol; The binding solution includes 60% formamide, 250mM NaH2PO4, 200mM NaCl, and 15mM sodium citrate solution, pH=7.0; 60% formamide acts as a heat stabilizer to stabilize the structure of DNA; The enzyme digestion solution was SmaI, NcoI, AvaI, and BspQI, with a concentration of 5 U / μL; The washing solution was phosphate buffered saline (PBS), pH = 7.

4.

2. The kit for detecting Shigella by a marker method according to claim 1, wherein The steps for constructing the Marker graph are as follows: Determine the absorbance OD of the Shigella bacterial solution gradient dilution 600 ; (2) After gradient dilution, plate the plate and count the colony counts (CFUs), establish a linear relationship between absorbance and colony count, and determine the true concentration of the bacterial solution; (3) Preparation of Shigella spp. culture solutions of different concentrations: 10 1 , 10 2 , 10 3 , 10 4 cfu / mL; (4) Use the kit method to detect the fluorescence intensity of each concentration of bacterial solution, establish a standard curve corresponding to the fluorescence intensity and the number of colonies, and verify it by the dilution plate method to obtain a marker graph; where Y1 on the left side of the marker graph is the fluorescence intensity value, and Y2 is the bacterial concentration.

3. The kit for detecting Shigella by a marker method according to claim 1, wherein The preparation of the magnetic aptamer comprises the following steps: Take 50 μL of 20 mg / mL SA magnetic beads in a 1.5 mL centrifuge tube, add 450 μL of buffer I, vortex and mix, then place on a magnetic stand for magnetic separation and discard the supernatant; the buffer I contains 2.5 mM Tris-HCl, 1.0 mM EDTA, 1.0 M NaCl, pH = 7.5; In step Buffer II was added to the centrifuge tube and the SA magnetic beads were mixed to a final concentration of 2 mg / mL; the buffer II contained 5.0 mM Tris-HCl, 2.0 mM EDTA, 2.0 M NaCl, pH = 7.5; Add 50 μL of specific aptamer Biotin-poly(dT)-ssDNA and rotate at room temperature for 30-60 minutes; After magnetic separation, the supernatant was discarded, and the mixture was washed 2-3 times with buffer solution. 100 μL of buffer solution I was added and mixed to obtain the magnetic aptamer.

4. The kit for detecting Shigella by a marker method according to claim 1, wherein The detection probes are labeled with fluorescein FITC, phycoerythrin PE, tetramethylrhodamine azide TAMRA-N, and tetramethylrhodamine isothiocyanate TRITC, with a concentration of 50 mM.

5. A method for detecting Shigella using a marker method, characterized in that: The following steps are involved: (1) Take 10 mL of the sample to be tested and filter it through an ultrafiltration membrane into a 1.5 mL centrifuge tube. Add 200 μL of bacterial lysis buffer and lyse it at room temperature for 10 min. Add 10 μL of enzyme digestion buffer and react at 37°C for 5 min. (2) Add 200 μL of binding solution and react at 65°C for 30-60 min. Add 50 μL of magnetic aptamer and 50 μL of detection aptamer and react at room temperature for 10 min. Separate the mixture with a magnetic stand and discard the supernatant. (3) Add 500 μL of washing solution and wash 2-3 times, then disperse in 100 μL of ddH2O to obtain magnetic aptamer- ipaH -Detect the aptamer complex; compare the fluorescence intensity with the marker image to determine the Shigella concentration of the sample to be tested.

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