A double probe reagent for detecting gladiolus Burkholderia and preparation method thereof

Abstract

The present invention relates to a dual-probe reagent for detecting gladiolus Burkholderia, belonging to the technical field of food detection. The dual-probe reagent is prepared by uniformly mixing a dual-probe composition and a reaction reagent. The dual-probe composition is prepared by uniformly mixing a magnetic separation probe and a yellow fluorescent nanozyme probe in a volume ratio of 1:1, the magnetic separation probe is a magnetic bead modified with streptavidin, and is obtained by specifically binding to an upstream primer SEA-F modified with biotin, and the yellow fluorescent nanozyme probe is obtained by amidation covalent coupling of an amino-containing yellow fluorescent nanozyme and a carboxyl-containing downstream primer SEA-R. The dual-probe reagent prepared by the present invention has a high degree of linear fitting, with a correlation coefficient as high as 0.99347, and presents a low detection limit as low as 13.67CFU / mL.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and particularly relates to a double-probe reagent for detecting Burkholderia gladiolus and a preparation method thereof. Background Art

[0002] Burkholderia gladioli is a foodborne pathogen widely distributed in nature. It survives in ecological niches such as soil, water and various animals, often coexists with fungi and plants, and has complex diversity within the species. Burkholderia gladioli has been isolated from patients with cystic fibrosis and is one of the three major bacteria causing cystic fibrosis in the United States. Cases of multiple skin abscesses associated with bacteremia caused by gladioli have also occurred in the elderly. In addition, Burkholderia gladioli has also been found to cause respiratory infections in patients with chronic granulomatous disease and cystic fibrosis. More seriously, Burkholderia gladioli can produce the human respiratory toxin fumonisin. Severe fumonisin poisoning can rapidly develop from liver and kidney damage to systemic multiple organ failure, with a very high mortality rate. In Africa and Asia, casualties caused by eating edible fungi, fermented rice and flour products, etc. contaminated with Burkholderia gladioli are common.

[0003] At present, conventional detection methods for this bacterium include traditional detection, molecular biological monitoring and immunological detection. These detection methods face two dilemmas: First, as a traditional detection method, microbial culture method requires multiple steps such as bacterial isolation and culture, morphological observation, and biochemical identification, which is cumbersome and time-consuming and does not meet the market demand for some rapid tests; second, molecular biological detection and immunological methods rely on equipment, and the specific recognition original antibodies have poor stability and high cost, making it difficult to promote and use them on an industrial scale. Therefore, it is urgent to develop a simple, rapid, highly sensitive and accurate detection method for real-time detection of gladiolus Burkholderia.

[0004] The detection method of emerging fluorescent biological probes is widely used in the determination of target substances due to its advantages of low cost, simple operation, fast response and high stability. At present, the main fluorescent markers used as detection probes include organic fluorescent dyes and quantum dots. Among them, traditional organic fluorescent dyes have low brightness and serious photobleaching problems, which hinder their practical application. In addition, the application of quantum dots is limited due to the shortcomings of cumbersome synthesis steps, poor stability, and incompatibility with various commercial media. Relatively speaking, carbon dots, also known as carbon nanodots or carbon quantum dots, are a type of zero-dimensional carbon nanomaterials with excellent fluorescence properties. They can be used as a potential non-toxic substitute for semiconductor quantum dots. Because single-mode detection has problems such as large matrix interference, poor signal accuracy, and single detection signal. Summary of the invention

[0005] In order to solve the problems of time-consuming and susceptibility to sample matrix in the detection of Burkholderia gladioli in food, the present invention provides a double-probe reagent for detecting Burkholderia gladioli, and also provides a method for preparing the double-probe reagent.

[0006] A 240 μL dual-probe reagent for detecting gladiolus Burkholderia is prepared by uniformly mixing 70 μL of the dual-probe composition and 170 μL of a reaction reagent;

[0007] The dual-probe composition is prepared by uniformly mixing a magnetic separation probe and a yellow fluorescent nanozyme probe in a volume ratio of 1:1;

[0008] The magnetic separation probe is a magnetic bead modified with streptavidin, and is obtained by specifically binding to the upstream primer SEA-F modified with biotin;

[0009] The yellow fluorescent nanozyme probe is obtained by covalently coupling an amino-containing yellow fluorescent nanozyme with a carboxyl-containing downstream primer SEA-R through amidation;

[0010] The reaction reagent is obtained by mixing 25 μL of 10×Bst reaction buffer, 5 μL of Bst DNA polymerase with an enzyme activity unit of 8 U / μL, 5 μL of 10 mM deoxyribonucleoside triphosphate (dNTP), 25 μL of 10% polyethylene glycol-200 and 110 μL of sterile double distilled water;

[0011] The upstream primer SEA-F: 5′-biotin-ATGCCGCCAATATCATCCG-3′,

[0012] The downstream primer SEA-R: 5′-carboxyl-CGAACCTTGCCCCGAGAA-3′.

[0013] The preparation steps of the dual probe reagent for detecting Burkholderia gladiolus are as follows:

[0014] (1) Preparation of activation mixture

[0015] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride with a molar concentration of 10 mM and N-hydroxysuccinimide with a molar concentration of 5 mM were mixed at a volume ratio of 1:1 to obtain an activated mixed solution.

[0016] (2) Preparation of magnetic separation probe

[0017] 20 μL of magnetic beads modified with streptavidin at a mass volume concentration of 500 μg / mL and 15 μL of upstream primer SEA-F at a molar concentration of 10 μM were vortexed and incubated at 4° C. for 3 h to prepare a magnetic separation probe.

[0018] (3) Preparation of yellow fluorescent nanozyme probe

[0019] 15 μL of 10 μM downstream primer SEA-R was vortexed and mixed with 15 μL of activation mixture, and incubated on a shaker at 200 rpm and room temperature for 30 min; then 5 μL of 5 mg / mL yellow fluorescent nanozyme was added and vortexed, and incubated on a shaker at 200 rpm and room temperature for 1 h to obtain a yellow fluorescent nanozyme probe.

[0020] (4) Preparation of dual probe reagents

[0021] At room temperature, 25 μL of 10×Bst reaction buffer, 5 μL of Bst DNA polymerase with an enzyme activity unit of 8U / μL, 5 μL of 10mM deoxyribonucleoside triphosphate (dNTP), 25 μL of 10% volume concentration of polyethylene glycol-200, 70 μL of dual probe composition and 110 μL of sterile double distilled water were mixed evenly to prepare 240 μL of dual probe reagent. The dual probe composition was prepared by mixing the magnetic separation probe and the yellow fluorescent nanozyme probe evenly in a volume ratio of 1:1.

[0022] The further technical solution is as follows:

[0023] In step (2), the preparation of the magnetic beads modified with streptavidin is performed as follows:

[0024] (2.1) Dissolve 21.6 mg of ferric chloride hexahydrate and 13.4 mg of sodium citrate in 20 mL of ethylene glycol, slowly add 23.0 mg of sodium acetate and stir vigorously for 30 min; incubate in a reactor under an oil bath at 220° C. for 10 h, cool naturally to room temperature to obtain carboxylated magnetic nanoparticles, wash the obtained carboxylated magnetic nanoparticles twice with ethanol and deionized water, and finally dilute with deionized water to 10 mg / mL to obtain a carboxylated magnetic nanoparticle suspension;

[0025] (2.2) washing 100 μL of the carboxylated magnetic nanoparticle suspension three times with 1×PBS buffer at a pH of 7.4, adding 100 μL of the activation mixture, and incubating in a blood mixer at room temperature, in the dark, and at a speed of 20 rpm for 30 min, washing twice with 1×PBS buffer at a pH of 7.4, and resuspending to 100 μL to obtain an activated carboxylated magnetic nanoparticle suspension;

[0026] (2.3) Slowly add 5 μL of 5 mg / mL streptavidin to the above 100 μL activated carboxylated magnetic nanoparticle suspension, and react at room temperature and 20 rpm for 3 h; add 5% BSA blocking solution and seal for 1 h, and wash 3 times with 1× PBS buffer at pH 7.4 to obtain magnetic beads modified with streptavidin.

[0027] In step (3), the preparation method of the yellow fluorescent nanozyme is as follows:

[0028] (3.1) 30 mL of 67 mM o-phenylenediamine was sonicated for 10 min, heated at 160 °C for 4 h in a polytetrafluoroethylene-lined reactor, and naturally cooled to room temperature; centrifuged at 10,000 rpm for 15 min to remove impurities; dialyzed in a 3,000 Da cellulose dialysis membrane for 24 h; and freeze-dried for 24 h to obtain yellow fluorescent carbon dots;

[0029] (3.2) Take 5 mL of yellow fluorescent carbon dots with a mass volume concentration of 1 mg / mL, add 1 mL of hexachloroplatinic acid hexahydrate with a molar concentration of 7.9 mM and shake evenly, add 1 mL of 0.1 M sodium borohydride during the ultrasonic process, and continue ultrasonication for 3 h; centrifuge at a speed of 10000 rpm for 30 min, discard the supernatant, resuspend with 20 mL of deionized water, and dialyze on a cellulose dialysis membrane with a molecular weight of 3000 Da for 24 h; freeze-dry for 24 h to obtain yellow fluorescent nanozyme.

[0030] The beneficial technical effects of the present invention are embodied in the following aspects:

[0031] 1. Through the visualization of the results, the positive control gladiolus Burkholderia DNA color development results showed that the characteristic ladder-shaped bands based on strand exchange amplification (SEA) mediated by denaturing vesicles appeared in the agarose gel electrophoresis detection, and the negative control sterile double distilled water color development results showed that no amplification bands appeared in the agarose gel electrophoresis, indicating that the primers SEA-F and SEA-R have good feasibility.

[0032] 2. It has been verified that the dual-probe reagent prepared by the present invention has a high degree of linear fitting, with a correlation coefficient as high as 0.99347, and presents a low detection limit of 13.67 CFU / mL. The detection limit of the existing technology for Burkholderia gladiolus is generally more than 10 2 CFU / mL; at the same time, the detection time of traditional technology varies from 6h to 7d, while the present invention adopts three modes for comparison detection, and the detection time can be controlled within 1h and has good specificity. In addition, the detection of the three-modal signal of colorimetric signal-fluorescence signal-photothermal signal, according to the obtained ultraviolet absorption intensity value-fluorescence intensity value-photothermal temperature value, is substituted into the obtained linear regression equation to obtain the concentration of gladiolus Burkholderia in the real sample. The three-modal signals verify each other, play an internal proofreading role, and improve the accuracy of the detection results.

[0033] 3. After testing, the linear regression equation established in the standard environment of the present invention can be directly applied to the detection of soaked black fungus samples. There is no need to re-cultivate bacteria to establish a standard curve. It is only necessary to directly apply the established linear regression equation, which saves time and is suitable for quickly estimating the preliminary concentration of gladiolus Burkholderia in food samples under emergency conditions. For example, the test results show that after calculation, in the environment of artificial inoculation of gladiolus Burkholderia in soaked black fungus, the measured concentrations of gladiolus Burkholderia are 1.07×10 7 CFU / mL, 1.07×10 7 CFU / mL, 0.94×10 7 CFU / mL (colorimetric-fluorescent-photothermal), and its actual concentration 10 7 The CFU / mL are close. It can be seen that even if the corresponding standard curve and linear regression equation are not established under the soaked black fungus sample environment, the standard curve and linear regression equation established under PBS buffer solution can still have good versatility. It can be seen that the yellow fluorescent nanozyme probe prepared by the present invention has good versatility and can be quickly applied to different sample detections.

[0034] 4. In summary, the present invention applies SEA technology to the recognition and detection of Burkholderia gladiolus for the first time, and assembles a magnetic separation probe through specific recognition between the upstream primer SEA-F modified with biotin and the magnetic beads modified with streptavidin. At the same time, the amino-rich yellow fluorescent nanozyme synthesized for the first time and the carboxyl-modified downstream primer SEA-R are covalently coupled through amidation to assemble a yellow fluorescent nanozyme probe. The above process effectively improves the sensitivity of detection; after magnetic absorption, the supernatant is used to simultaneously detect the fluorescence signal and the colorimetric signal, and then the resuspended precipitate after the colorimetric signal detection is subjected to photothermal signal detection. The colorimetric-fluorescence-photothermal triple-modal signals can be obtained simultaneously in one experiment, which not only enhances the sensitivity of detection, but also can correct each other to avoid misjudgment caused by operational errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is an agarose gel electrophoresis diagram of Burkholderia gladiolus SEA in the present invention;

[0036] Figure 2 is a transmission electron microscope image of magnetic beads modified with streptavidin prepared in the present invention;

[0037] Figure 3 is the X-ray photoelectron spectrum of the yellow fluorescent nanozyme prepared in the present invention;

[0038] Figure 4 The ultraviolet absorption graph of different concentrations of Burkholderia gladiolus in the present invention and the corresponding standard curve graph;

[0039] Figure 5 The fluorescence spectra of different concentrations of Burkholderia gladiolus in the present invention and the corresponding standard curve diagram;

[0040] Figure 6 is a standard curve diagram of the photothermal temperature values ​​of different concentrations of Burkholderia gladiolus in the present invention;

[0041] Figure 7 This is a specific detection diagram of Burkholderia gladiolus in the present invention. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments.

[0043] Unless otherwise defined, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] The raw materials used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; the % in the following examples are all mass percentages unless otherwise specified.

[0045] In the following examples, the upstream primer SEA-F: 5′-biotin-ATGCCGCCAATATCATCCG-3′ and the downstream primer SEA-R: 5′-carboxyl-CGAACCTTGCCCCGAGAA-3′ were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the hexachloroplatinic acid hexahydrate, o-phenylenediamine, and sodium borohydride were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; unless otherwise specified, other raw materials were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0046] Among the strains used in the present invention, the number of Burkholderia gladiolus is ATCC 33664, the number of Listeria monocytogenes is ATCC 43251, the number of Staphylococcus aureus is ATCC 29213, the number of Salmonella is ATCC 14028, the number of Escherichia coli is ATCC 25922, the number of Cronobacter sakazakii is ATCC 29544, the number of Pseudomonas aeruginosa is ATCC 15442, and the number of Vibrio parahaemolyticus is ATCC17802.

[0047] The instruments, equipment, raw materials, reagents or method steps used in the present invention are all ensured to be processed under sterile conditions.

[0048] The instruments, equipment, raw materials, reagents or method steps not mentioned in the present invention are conventional or well-known technical methods for those skilled in the art and will not be described in detail in the present invention.

[0049] Example 1

[0050] The preparation steps of the dual probe reagent for detecting Burkholderia gladiolus are as follows:

[0051] (1) Preparation of activation mixture

[0052] Mix 10 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mM N-hydroxysuccinimide at a volume ratio of 1:1 to obtain an activated mixed solution, which is stored in a refrigerator at 4°C for later use.

[0053] (2) Preparation of magnetic separation probe

[0054] 20 μL of magnetic beads modified with streptavidin at a mass volume concentration of 500 μg / mL and 15 μL of upstream primer SEA-F at a molar concentration of 10 μM were vortexed and incubated at 4° C. for 3 h to prepare a magnetic separation probe.

[0055] Upstream primer SEA-F: 5′-biotin-ATGCCGCCAATATCATCCG-3′.

[0056] The preparation of magnetic beads modified with streptavidin is as follows:

[0057] (2.1) Dissolve 21.6 mg of ferric chloride hexahydrate and 13.4 mg of sodium citrate in 20 mL of ethylene glycol, slowly add 23.0 mg of sodium acetate and stir vigorously for 30 min; incubate in a reactor under an oil bath at 220°C for 10 h, cool naturally to room temperature to obtain carboxylated magnetic nanoparticles, wash the obtained carboxylated magnetic nanoparticles twice with ethanol and deionized water, and finally dilute with deionized water to 10 mg / mL to obtain a carboxylated magnetic nanoparticle suspension.

[0058] (2.2) washing 100 μL of the carboxylated magnetic nanoparticle suspension three times with 1×PBS buffer at a pH of 7.4, adding 100 μL of the activation mixture, and incubating in a blood mixer at room temperature, in the dark, and at a speed of 20 rpm for 30 min, washing twice with 1×PBS buffer at a pH of 7.4, and resuspending to 100 μL to obtain an activated carboxylated magnetic nanoparticle suspension;

[0059] (2.3) Slowly add 5 μL of 5 mg / mL streptavidin to the above 100 μL activated carboxylated magnetic nanoparticle suspension, react at room temperature and 20 rpm for 3 h; add 5% BSA blocking solution and seal for 1 h, wash three times with 1× PBS buffer with a pH value of 7.4 to obtain magnetic beads modified with streptavidin. Transmission electron microscopy images of magnetic beads modified with streptavidin, as shown in Figure 2 shown.

[0060] (3) Preparation of yellow fluorescent nanozyme probe

[0061] 15 μL of 10 μM downstream primer SEA-R was vortexed and mixed with 15 μL of activation mixture, and incubated on a shaker at 200 rpm and room temperature for 30 min; then 5 μL of 5 mg / mL yellow fluorescent nanozyme was added and vortexed, and incubated on a shaker at 200 rpm and room temperature for 1 h to obtain a yellow fluorescent nanozyme probe.

[0062] Downstream primer SEA-R: 5′-Carboxy-CGAACCTTGCCCCGAGAA-3′.

[0063] When the ZETA potential of the yellow fluorescent nanozyme dropped from +5.50 mV to -28.17 mV, it indicated that the primer SEA-R in the yellow fluorescent nanozyme probe was successfully modified onto the yellow fluorescent nanozyme.

[0064] The yellow fluorescent nanozyme probe is obtained by amidation and covalent coupling of an amino-containing yellow fluorescent nanozyme and a carboxyl-containing downstream primer SEA-R.

[0065] The preparation procedure of yellow fluorescent nanozyme is as follows:

[0066] (3.1) 30 mL of 67 mM o-phenylenediamine was sonicated for 10 min, heated at 160 °C for 4 h in a polytetrafluoroethylene-lined reactor, and naturally cooled to room temperature; centrifuged at 10,000 rpm for 15 min to remove impurities; dialyzed in a 3,000 Da cellulose dialysis membrane for 24 h; and freeze-dried for 24 h to obtain yellow fluorescent carbon dots;

[0067] (3.2) Take 5 mL of yellow fluorescent carbon dots with a mass volume concentration of 1 mg / mL, add 1 mL of hexachloroplatinic acid hexahydrate with a molar concentration of 7.9 mM and shake evenly, add 1 mL of 0.1 M sodium borohydride during the ultrasonic process, and continue ultrasonication for 3 h; centrifuge at a speed of 10000 rpm for 30 min, discard the supernatant, resuspend with 20 mL of deionized water, and dialyze on a cellulose dialysis membrane with a molecular weight of 3000 Da for 24 h; freeze-dry for 24 h to obtain yellow fluorescent nanozyme.

[0068] See also Figure 3 In the X-ray photoelectron spectrum of the yellow fluorescent nanozyme, the main elements present in the yellow fluorescent nanozyme are platinum (Pt), carbon (C), nitrogen (N), and oxygen (O), indicating that platinum (Pt) was successfully incorporated into the yellow fluorescent nanozyme.

[0069] (4) Preparation of dual probe reagents

[0070] At room temperature, 25 μL of 10×Bst reaction buffer, 5 μL of Bst DNA polymerase with an enzyme activity unit of 8 U / μL, 5 μL of 10 mM deoxyribonucleoside triphosphate (dNTP), 25 μL of 10% volume concentration of polyethylene glycol-200, 70 μL of dual probe composition and 110 μL of sterile double distilled water were mixed evenly to prepare 240 μL of dual probe reagent.

[0071] The dual-probe composition is prepared by uniformly mixing a magnetic separation probe and a yellow fluorescent nanozyme probe in a volume ratio of 1:1.

[0072] When the target detection object Burkholderia gladioli is present, a colorimetric signal, a fluorescence signal and a photothermal signal that are dependent on the concentration of Burkholderia gladioli are generated.

[0073] Example 2

[0074] Visual detection of Burkholderia gladiolus using SEA primers:

[0075] (1) SEA primers were used to amplify the H. gladiolus strain

[0076] ① The present invention uses the gene bonJ (GenBank: AFN27476.1) of gladiolus Burkholderia ATCC 33664 as a reference sequence to design SEA detection primers with specific amplification effect on gladiolus Burkholderia, including a pair of primers (SEA-F and SEA-R), wherein SEA-F: 5′-biotin-ATGCCGCCAATATCATCCG-3′; SEA-R: 5′-carboxyl-CGAACCTTGCCCCGAGAA-3′. The 250μL SEA reaction system contains 25μL 10×Bst reaction buffer, 5μL Bst DNA polymerase with an enzyme activity unit of 8U / μL, 5μL 10mM deoxyribonucleoside triphosphate (dNTP), 25μL 10% polyethylene glycol-200, 15μL 10μM upstream primer SEA-F, 15μL 10μM downstream primer SEA-R and 10μL extracted DNA template. The insufficient part is supplemented with sterile double distilled water; the SEA reaction conditions are incubated in a 61℃ water bath for 30min. Use 10μL sterile double distilled water without target gene for control amplification to replace DNA template. ② After the SEA reaction, take 10μL of amplified product and detect it by 2% agarose gel electrophoresis. If the characteristic ladder band of SEA appears, it is judged as positive, and if there is no amplification band, it is judged as negative.

[0077] (2) Visualization of test results: The positive control gladiolus Burkholderia DNA color development results showed that the characteristic ladder-shaped bands of SEA appeared in agarose gel electrophoresis, while the negative control sterile double distilled water color development results showed that no amplified bands appeared in agarose gel electrophoresis. Figure 1 The lanes are marked with DL 500 DNA Marker, a DNA molecular weight standard for electrophoresis. Lane 1 is the positive control Burkholderia gladioli DNA, and lane 2 is the negative control sterile double distilled water, indicating that this primer has good feasibility.

[0078] Example 3

[0079] Construction of linear regression equation for colorimetric-fluorescence-photothermal trimodal signal detection of Burkholderia gladiolus

[0080] The prepared dual-probe reagent was used to establish a linear regression equation for detecting Burkholderia gladiolus. The operation steps are as follows:

[0081] (1) Pretreatment

[0082] First, a test solution of a non-bacterial test sample was prepared, and the test solution was artificially contaminated with Burkholderia gladioli, and a linear gradient dilution of the bacterial solution was set; the concentrations of 10 0 CFU / mL, 101 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL bacterial solution; extract and dilute the bacterial solution into different gradients for later use.

[0083] (2) Detection of colorimetric signals

[0084] 50 μL of the supernatant obtained after magnetic separation was taken, and 420 μL of acetic acid-sodium acetate buffer was added, wherein the acetic acid-sodium acetate buffer was as follows: acetic acid-sodium acetate buffer with a pH value of 4.5 was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and its product number was R27435-500ml, and 3,3',5,5'-tetramethylbenzidine with a concentration of 1 mM and a hydrogen peroxide solution with a concentration of 0.2 mM were added, and the mixture was reacted at 35°C for 10 min to obtain a colorimetric signal detection solution, and an ultraviolet spectrophotometer was used to detect the ultraviolet absorption intensity value generated at 653 nm, and the read ultraviolet absorption intensity value was selected as the ordinate, and the logarithmic value of the bacterial concentration of the test solution was used as the abscissa to draw a standard curve, and the correlation coefficient and the detection limit were obtained according to the linear regression equation; Figure 4 As shown, see Figure 4 A in the figure, from top to bottom, are: Curve 1 represents a bacterial concentration of 10 0 The UV absorption curve of CFU / mL, curve 2 indicates that the bacterial concentration is 10 1 The UV absorption curve of CFU / mL, curve 3 indicates that the bacterial concentration is 10 2 The UV absorption curve of CFU / mL, curve 4 indicates that the bacterial concentration is 10 3 The UV absorption curve of CFU / mL, curve 5 indicates that the bacterial concentration is 10 4 The UV absorption curve of CFU / mL, curve 6 indicates that the bacterial concentration is 10 5 The UV absorption curve of CFU / mL, curve 7 indicates that the bacterial concentration is 10 6 The UV absorption curve of CFU / mL, curve 8 indicates that the bacterial concentration is 10 7 The UV absorption curve of CFU / mL, curve 9 indicates that the bacterial concentration is 10 8 The UV absorption curve of CFU / mL, curve 10 indicates that the bacterial concentration is 10 9 CFU / mL UV absorption curve; at the same time, combined with Figure 4 From the perspective of B in the above Figure 4 The ultraviolet absorption intensity value at 653 nm on each ultraviolet absorption curve in A is taken as the ordinate, and the logarithmic value of the bacterial concentration corresponding to each ultraviolet absorption curve is taken as the abscissa. The linear regression equation is y = -0.13425x + 1.15007, and the detection limit is 13.67 CFU / mL. 2 The value is 0.99111.

[0085] (3) Detection of fluorescence signals

[0086] The supernatant obtained by magnetic separation was used to obtain a fluorescent signal detection solution. The fluorescence intensity value generated at an excitation wavelength of 440 nm was detected using a fluorescence spectrophotometer. The read fluorescence intensity value was selected as the ordinate, and the logarithm of the bacterial concentration of the detected solution was used as the abscissa to draw a standard curve. The correlation coefficient and the detection limit were obtained according to the linear regression equation; Figure 5 As shown, see Figure 5 A in the figure, from top to bottom, are: Curve 1 represents a bacterial concentration of 10 0 The fluorescence spectrum curve of CFU / mL (blank control test solution) and curve 2 indicate that the bacterial concentration is 10 1 The fluorescence spectrum curve of CFU / mL, curve 3 indicates that the bacterial concentration is 10 2 The fluorescence spectrum curve of CFU / mL, curve 4 indicates that the bacterial concentration is 10 3 The fluorescence spectrum curve of CFU / mL, curve 5 indicates that the bacterial concentration is 10 4 The fluorescence spectrum curve of CFU / mL, curve 6 indicates that the bacterial concentration is 10 5 The fluorescence spectrum curve of CFU / mL, curve 7 indicates that the bacterial concentration is 10 6 The fluorescence spectrum curve of CFU / mL, curve 8 indicates that the bacterial concentration is 10 7 The fluorescence spectrum curve of CFU / mL, curve 9 indicates that the bacterial concentration is 10 8 The fluorescence spectrum curve of CFU / mL, curve 10 indicates that the bacterial concentration is 10 9 CFU / mL fluorescence spectrum curve; at the same time, combined with Figure 5 From the perspective of B in the above Figure 5 The fluorescence intensity value at 567 nm on each fluorescence spectrum curve in A is taken as the ordinate, and the logarithm of the bacterial concentration corresponding to each fluorescence spectrum curve is taken as the abscissa. The linear regression equation is y = -0.13778x + 1.04605, and the detection limit is 15.55 CFU / mL. 2 The value is 0.99347.

[0087] (4) Detection of photothermal signals

[0088] In addition, the above colorimetric signal detection solution was directly used as a photothermal signal detection solution at 1.5 W / cm 2 The 660nm laser was irradiated for 120s, and the infrared thermal imager was used to take the thermal image of the photothermal detection solution temperature. The photothermal temperature value read was selected as the ordinate, and the logarithm of the bacterial concentration of the detected solution was used as the abscissa to draw the standard curve. The correlation coefficient and detection limit were obtained according to the linear regression equation. Figure 6 As shown in the figure, the temperature values ​​corresponding to different concentrations of gladiolus Burkholderia are used as the ordinate, and the logarithm of the corresponding bacterial concentration is used as the abscissa. The linear regression equation is y=-2.82008x+41.07007, and the detection limit is 19.98 CFU / mL. 2 The value is 0.98355.

[0089] Example 4

[0090] Testing of real samples

[0091] The dual-probe reagent prepared in Example 1 and the linear regression equation constructed in Example 3 were used to detect real food samples, and the soaked black fungus samples were selected for testing:

[0092] In order to verify whether the linear regression equation constructed according to Example 3 can be effectively applied to the determination under the interference environment (soaked black fungus), this Example 4 is artificially quantitatively infected to explore the difference between the number of bacteria detected by the dual-probe composition in the soaked black fungus sample, so as to find out that even in real food samples, the dual-probe composition established in this application can be effectively detected under the influence of actual samples with nutrients.

[0093] The soaked black fungus was inoculated with Burkholderia gladiolus, and the inoculation amount was 10 7 CFU / mL, the spiked black fungus was homogenized by a homogenizer for 2 minutes, allowed to stand for 5 minutes, and the supernatant was taken as the test solution. In combination with Example 3, the colorimetric-fluorescence-photothermal trimodal signal detection was performed, and the obtained ultraviolet absorption intensity value-fluorescence intensity value-photothermal temperature value was substituted into the obtained linear regression equation to obtain the concentration of gladiolus Burkholderia in the real sample.

[0094] The experimental results show that, according to calculation, in the environment of artificial inoculation of gladiolus Burkholderia in soaked black fungus, the concentrations of gladiolus Burkholderia were 1.07×10 7 CFU / mL, 1.07×10 7 CFU / mL, 0.94×10 7 CFU / mL (colorimetric-fluorescent-photothermal), and its actual concentration 10 7The CFU / mL are close. It can be seen that even if the corresponding standard curve and linear regression equation are not established under the soaked black fungus sample environment, the standard curve and linear regression equation established under PBS buffer solution can still have good versatility. It can be seen that the yellow fluorescent nanozyme probe prepared by the present invention has good versatility and can be quickly applied to different sample detections.

[0095] Example 5

[0096] Basically the same as Example 3, except that:

[0097] The gladiolus Burkholderia was replaced with Listeria monocytogenes, Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, Pseudomonas aeruginosa, and Vibrio parahaemolyticus; the concentrations of each were 10 8 CFU / mL of the test solution containing bacteria was then used to extract DNA.

[0098] See also Figure 7 A in the figure, the test solution after adding Burkholderia gladiolus (sequence number "1" in the figure), Listeria monocytogenes (sequence number "2" in the figure), Staphylococcus aureus (sequence number "3" in the figure), Salmonella (sequence number "4" in the figure), Escherichia coli (sequence number "5" in the figure), Cronobacter sakazakii (sequence number "6" in the figure), Pseudomonas aeruginosa (sequence number "7" in the figure), and Vibrio parahaemolyticus (sequence number "8" in the figure), the Listeria monocytogenes The ultraviolet absorption intensity value is 0.97362, the ultraviolet absorption intensity value of Staphylococcus aureus is 0.97519, the ultraviolet absorption intensity value of Salmonella is 0.97086, the ultraviolet absorption intensity value of Escherichia coli is 0.98110, the ultraviolet absorption intensity value of Cronobacter sakazakii is 0.98031, the ultraviolet absorption intensity value of Pseudomonas aeruginosa is 0.98661, and the ultraviolet absorption intensity value of Vibrio parahaemolyticus is 0.99999. At the same time, the ultraviolet absorption intensity value of the test solution after adding gladiolus Burkholderia (serial number "1" in the figure) under the same conditions is 0.067.

[0099] See also Figure 7In B, the test solution after adding Burkholderia gladiolus (sequence number "1" in the figure), Listeria monocytogenes (sequence number "2" in the figure), Staphylococcus aureus (sequence number "3" in the figure), Salmonella (sequence number "4" in the figure), Escherichia coli (sequence number "5" in the figure), Cronobacter sakazakii (sequence number "6" in the figure), Pseudomonas aeruginosa (sequence number "7" in the figure), and Vibrio parahaemolyticus (sequence number "8" in the figure), the fluorescence intensity value of Listeria monocytogenes is 0.94673, the fluorescence intensity value of Staphylococcus aureus is 0.97987, the fluorescence intensity value of Salmonella is 0.99998, the fluorescence intensity value of Escherichia coli is 0.95107, the fluorescence intensity value of Cronobacter sakazakii is 0.97708, the fluorescence intensity value of Pseudomonas aeruginosa is 0.98204, and the fluorescence intensity value of Vibrio parahaemolyticus is 0.98792. At the same time, under the same conditions, the test solution after adding Burkholderia gladiolus (serial number "1" in the figure) had a fluorescence intensity value of 0.10697.

[0100] See also Figure 7 C in the figure, the test solution after adding Burkholderia gladiolus (serial number "1" in the figure), Listeria monocytogenes (serial number "2" in the figure), Staphylococcus aureus (serial number "3" in the figure), Salmonella (serial number "4" in the figure), Escherichia coli (serial number "5" in the figure), Cronobacter sakazakii (serial number "6" in the figure), Pseudomonas aeruginosa (serial number "7" in the figure), and Vibrio parahaemolyticus (serial number "8" in the figure), the photothermal temperature value of Listeria monocytogenes is 40.17, the photothermal temperature value of Staphylococcus aureus is 39.97, the photothermal temperature value of Salmonella is 40.83, the photothermal temperature value of Escherichia coli is 40.47, the photothermal temperature value of Cronobacter sakazakii is 40.43, the photothermal temperature value of Pseudomonas aeruginosa is 40.60, and the photothermal temperature value of Vibrio parahaemolyticus is 41.17. At the same time, under the same conditions, the photothermal temperature of the test solution after adding Burkholderia gladiolus (serial number "1" in the figure) was 21.33.

[0101] This indicates that the dual-probe composition prepared in the present invention has good specificity for Burkholderia gladiolus.

[0102] It will be easily understood by those skilled in the art that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dual-probe reagent for detecting Burkholderia gladiolus, characterized in that: 240 μL of the dual probe reagent is prepared by uniformly mixing 70 μL of the dual probe composition and 170 μL of the reaction reagent; The dual-probe composition is prepared by uniformly mixing a magnetic separation probe and a yellow fluorescent nanozyme probe in a volume ratio of 1:1; The magnetic separation probe is a magnetic bead modified with streptavidin, and is obtained by specifically binding to the upstream primer SEA-F modified with biotin; The yellow fluorescent nanozyme probe is obtained by amidation and covalent coupling of an amino-containing yellow fluorescent nanozyme and a carboxyl-containing downstream primer SEA-R; The preparation method of the yellow fluorescent nanozyme is as follows: 30 mL of o-phenylenediamine with a molar concentration of 67 mM was ultrasonically treated for 10 min, heated at 160°C for 4 h in a reactor lined with polytetrafluoroethylene, and naturally cooled to room temperature; centrifuged at 10,000 rpm for 15 min to remove impurities; dialyzed in a 3,000 Da cellulose dialysis membrane for 24 h; and freeze-dried for 24 h to obtain yellow fluorescent carbon dots; Take 5 mL of yellow fluorescent carbon dots with a mass volume concentration of 1 mg / mL, add 1 mL of hexachloroplatinic acid hexahydrate with a molar concentration of 7.9 mM and shake evenly, add 1 mL of 0.1 M sodium borohydride during the ultrasonic process, and continue ultrasonication for 3 hours; centrifuge at a speed of 10000 rpm for 30 minutes, discard the supernatant, resuspend with 20 mL of deionized water, and dialyze on a cellulose dialysis membrane with a molecular weight of 3000 Da for 24 hours; freeze-dry for 24 hours to obtain yellow fluorescent nanozyme; The reaction reagent is obtained by mixing 25 μL of 10×Bst reaction buffer, 5 μL of Bst DNA polymerase with an enzyme activity unit of 8 U / μL, 5 μL of deoxyribonucleoside triphosphate with a molar concentration of 10 mM, 25 μL of polyethylene glycol-200 with a volume concentration of 10%, and 110 μL of sterile double distilled water; The upstream primer SEA-F: 5′-biotin-ATGCCGCCAATATCATCCG-3′, The downstream primer SEA-R: 5′-carboxyl-CGAACCTTGCCCCGAGAA-3′.

2. A method for preparing a dual-probe reagent for detecting Burkholderia gladiolus, characterized in that: The preparation steps are as follows: (1) Preparation of activation mixture Mix 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride having a molar concentration of 10 mM and N-hydroxysuccinimide having a molar concentration of 5 mM in a volume ratio of 1:1 to obtain an activated mixed solution; (2) Preparation of magnetic separation probe 20 µL of magnetic beads modified with streptavidin at a mass volume concentration of 500 µg / mL and 15 µL of upstream primer SEA-F at a molar concentration of 10 µM were vortexed and incubated at 4°C for 3 h to prepare a magnetic separation probe; The upstream primer SEA-F: 5′-biotin-ATGCCGCCAATATCATCCG-3′; (3) Preparation of yellow fluorescent nanozyme probe 15 µL of 10 µM downstream primer SEA-R and 15 µL of activation mixture were vortexed and incubated on a shaker at 200 rpm and room temperature for 30 min; 5 µL of 5 mg / mL yellow fluorescent nanozyme was then added and vortexed, and incubated on a shaker at 200 rpm and room temperature for 1 h to obtain a yellow fluorescent nanozyme probe; The downstream primer SEA-R: 5′-carboxyl-CGAACCTTGCCCCGAGAA-3′; In step (3), the preparation method of the yellow fluorescent nanozyme is as follows: (3.1) 30 mL of 67 mM o-phenylenediamine was sonicated for 10 min, heated at 160 °C for 4 h in a polytetrafluoroethylene-lined reactor, and naturally cooled to room temperature; centrifuged at 10,000 rpm for 15 min to remove impurities; dialyzed in a 3,000 Da cellulose dialysis membrane for 24 h; and freeze-dried for 24 h to obtain yellow fluorescent carbon dots; (3.2) Take 5 mL of yellow fluorescent carbon dots with a mass volume concentration of 1 mg / mL, add 1 mL of hexachloroplatinic acid hexahydrate with a molar concentration of 7.9 mM, shake evenly, add 1 mL of 0.1 M sodium borohydride during the ultrasonic process, and continue ultrasonication for 3 hours; centrifuge at a speed of 10000 rpm for 30 minutes, discard the supernatant, resuspend with 20 mL of deionized water, and dialyze on a cellulose dialysis membrane with a molecular weight of 3000 Da for 24 hours; freeze-dry for 24 hours to obtain yellow fluorescent nanozyme; (4) Preparation of dual probe reagents At room temperature, 25 μL of 10×Bst reaction buffer, 5 μL of Bst DNA polymerase with an enzyme activity unit of 8 U / μL, 5 μL of 10 mM deoxyribonucleoside triphosphate, 25 μL of 10% volume concentration of polyethylene glycol-200, 70 μL of dual probe composition and 110 μL of sterile double distilled water were mixed evenly to prepare 240 μL of dual probe reagent; The dual-probe composition is prepared by uniformly mixing a magnetic separation probe and a yellow fluorescent nanozyme probe in a volume ratio of 1:

1.

3. The preparation method according to claim 2, characterized in that: In step (2), the preparation of the magnetic beads modified with streptavidin is performed as follows: (2.1) Dissolve 21.6 mg of ferric chloride hexahydrate and 13.4 mg of sodium citrate in 20 mL of ethylene glycol, slowly add 23.0 mg of sodium acetate and stir vigorously for 30 min; incubate in a reactor under an oil bath at 220°C for 10 h, cool naturally to room temperature to obtain carboxylated magnetic nanoparticles, wash the obtained carboxylated magnetic nanoparticles twice with ethanol and deionized water, and finally dilute with deionized water to 10 mg / mL to obtain a carboxylated magnetic nanoparticle suspension; (2.2) Wash 100 μL of the carboxylated magnetic nanoparticle suspension three times with 1×PBS buffer at pH 7.4, add 100 μL of the activation mixture, and incubate in a blood mixer at room temperature, away from light, and at a speed of 20 rpm for 30 min, wash twice with 1×PBS buffer at pH 7.4, and resuspend to 100 μL to obtain an activated carboxylated magnetic nanoparticle suspension; (2.3) Slowly add 5 μL of 5 mg / mL streptavidin to 100 μL of the activated carboxylated magnetic nanoparticle suspension, and react at room temperature and 20 rpm for 3 h; add 5% BSA blocking solution and seal for 1 h, and wash three times with 1× PBS buffer at pH 7.4 to obtain magnetic beads modified with streptavidin.

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