CN107365836A - Method based on nucleic acid chromatography biosensor technique detection Bacillus cereus - Google Patents

Abstract

The invention relates to a method for detecting Bacillus cereus based on nucleic acid chromatography biosensing technology. According to the virulence gene hbl A of Bacillus cereus, a loop-mediated isothermal amplification primer (SEQ ID NO: 1‑4) is designed , combined with nanozyme nucleic acid test strips, to establish a detection method for Bacillus cereus based on LAMP nanozyme sensor. This method can be successfully used to distinguish live bacteria cells from dead bacteria cells, and the detection limit of Bacillus cereus can reach 10CFU/mL.

Description

Method for detecting Bacillus cereus based on nucleic acid chromatography biosensing technology

technical field

The invention relates to the technical field of biosensing detection, in particular to a method for detecting Bacillus cereus based on nucleic acid chromatography biosensing technology.

Background technique

Bacillus cereus, rod-shaped, round, colony diameter 2.5mm, raised, smooth surface, neat edges, translucent, creamy, light yellow, Gram stain negative, oxidase positive, contact enzyme positive , Negative amylase test, negative gelatin liquefaction test, oxidative fermentation test of glucose, no hydrogen sulfide, negative sulfate reduction test, producing water-soluble green pigment. Bacillus cereus is associated with a small number of food poisonings (approximately 2–5%), including some with severe nausea, vomiting, and abdominal pain. Roughly speaking, bacillary food poisoning is caused by incorrect cooking methods that leave bacterial spores on food, or worse, improper freezing of food that allows the spores to germinate. The result of bacterial reproduction is the production of enterotoxins, and people will have vomiting, diarrhea and other adverse symptoms after eating food containing toxins.

The traditional bacterial detection method is mainly based on physiological and biochemical characteristics, but the traditional detection method needs to go through steps such as pre-enrichment, selective plate separation, biochemical identification, etc. It takes 5-7 days from sampling to confirming the result, the detection cycle is long, and the operation is cumbersome , the workload is heavy; using the specificity of antigen-antibody reaction to identify bacteria has a history of more than half a century, but the screening of microbial antibodies is very cumbersome, and the final detection specificity is not high; molecular biology detection technology The continuous improvement and development of the traditional detection method has overcome the problems of cumbersome experimental operation and long time consumption, and has also led to the rapid development of rapid detection methods for microorganisms. However, the disadvantage of molecular biology methods is that it is not easy to analyze the results. With the development of modern biotechnology, biosensors, a new technology that is faster and more sensitive than traditional methods, have emerged in the field of food safety detection. Biosensors have the advantages of strong specificity and high sensitivity, which can simplify the analysis and detection steps, shorten the analysis time, and more importantly, make online real-time detection possible, easy to carry and field operations, and have developed rapidly in the field of food safety. In recent years, the development and application of many new materials and technologies are becoming research hotspots, injecting new vitality into the development of biosensors, and showing great application potential in the field of microbial detection. Therefore, it is of great significance to establish a reliable and rapid sensor detection method for Bacillus cereus, especially a method that can identify dead and alive bacteria, in daily monitoring and market screening.

Contents of the invention

The purpose of the present invention is to provide a method for detecting bacillus cereus based on nucleic acid chromatography biosensing technology.

The present invention is conceived as follows: to develop a rapid and ultra-sensitive detection method for Bacillus cereus, especially a method capable of identifying dead or alive bacteria, to establish a method based on propidium iodide azide (PMA), loop-mediated isothermal amplification reaction (LAMP) ) and a continuous cascade nanozyme biosensor for live bacteria detection of nanozyme test paper. The hbl A gene of Bacillus cereus was assayed using fluorescein (FITC)-modified and biotin (BIO)-modified primers in a LAMP reaction. The combination of PMA and LAMP was applied to the isolation of dead and alive Bacillus cereus. Then, use nanozyme probes to prepare magnetic particle-based immunochromatographic strips (nanozyme strips) for detecting amplification signals, and perform result interpretation or quantification by visual inspection or using a strip reader for on-site detection of wax samples Live Bacillus provides a fast, ultrasensitive and convenient tool.

In order to achieve the purpose of the present invention, the present invention firstly provides a LAMP primer set for detecting Bacillus cereus (Bacillus cereus), including (SEQ ID NO:1-4):

Outer forward primer F3: 5'-TGCTATTTTGGGTCTACCAA-3';

Outer reverse primer B3: 5'-GTGGACATATAAGTAAGAGCGTTA-3'; and

Inner forward primer FIP: 5'-ACGTAATTCTGCTAATAAAGGCTCTAATT GGCGGTATTATAGTCGG-3';

Inner reverse primer BIP: 5'-AACCTTAAAATCGTGTAGTTGGAGTTATCATCAAGCGCCTTGTG-3'.

Among them, the 5' end of the primer FIP was labeled with biotin, and the 5' end of the primer BIP was labeled with fluorescein FITC.

The present invention also provides a nanozyme nucleic acid chromatography test strip, and the preparation method of the test strip comprises the following steps:

1) Preparation of Fe ₃ O ₄ magnetic particles;

2) Preparation of nanozyme probe (MNP): incubate Fe ₃ O ₄ magnetic particles with biotin secondary antibody to obtain biotin secondary antibody nanozyme probe;

3) Assembly of nanozyme nucleic acid chromatography test strips: the test strips include a sample pad, a binding pad, a nitrocellulose membrane and an absorption pad, wherein the nitrocellulose membrane is provided with at least one detection line and 1 quality control line; the biotin secondary antibody nanozyme probe is immobilized on the binding pad;

① Use FITC antibody and biotin antibody to draw the detection line and quality control line on the nitrocellulose membrane respectively, and dry;

② Paste the above-mentioned sample pads, binding pads, nitrocellulose membranes with test lines and quality control lines, and absorbent pads on the bottom plate (plastic liner) in sequence to complete the assembly of the test strip. The schematic diagram of the assembled test strip is shown in Figure 5.

In step 1), Fe ₃ O ₄ magnetic particles were synthesized by hydrothermal method, specifically: dissolving 0.6-0.8g FeCl ₃ ·6H ₂ O in 20mL of ethylene glycol, then adding 1.5-2.0g sodium acetate, stirring vigorously for 30 -40min, then sealed in an autoclave, heated at 200°C for 16-18h; the magnetic particle product was washed several times with ethanol, and dried at 60°C; 5-8mg each of dichloroethane and N-hydroxysuccinimide Dissolve in 1 mL of deionized water by vortexing to prepare a mixed solution, then add 5-8 mg of magnetic particles into the mixed solution, incubate at room temperature for 30-40 min, then collect the magnetic particles with a magnet, wash twice with ultrapure water, that is Get Fe ₃ O ₄ magnetic particles.

Step 2) specifically: adding the biological antibody with a concentration of 100 μg/mL into 50 mM sodium acetate buffer solution with pH 6.0, and then mixing with 5-8 mg of Fe ₃ O ₄ magnetic particles, vortexing the mixture, and incubating at 4° C. overnight; The mixture was washed twice with PBS solution of pH 7.0, and then incubated in 50 mM Tris buffer solution of pH 7.2 for 30 min at room temperature; then washed with PBS solution of pH 7.0 to obtain the biotin secondary antibody nanozyme probe.

In step 3), the detection line is set at a position 1.1 cm away from the lower edge of the nitrocellulose membrane, and the quality control line is set at a position 1.6 cm away from the lower edge of the nitrocellulose membrane. The distance between the detection line and the quality control line is 4.5mm. Spray FITC antibody and biotin antibody on the test line and quality control line of the nitrocellulose membrane at 1.0 μL/cm, respectively. The concentration of the antibody coated on the detection line and the quality control line is both 0.5-2 mg/mL. The optimal concentration of antibody is 1mg/mL.

The biotin antibody used in the present invention was purchased from Sigma, B7653, and the FITC antibody was purchased from Sigma, F5636. The biotin secondary antibody was goat anti-mouse IgG. The nitrocellulose membrane used was Millipore135S.

In a preferred embodiment of the present invention, the preparation of the nanozyme nucleic acid chromatography test strip is as follows:

In step 1), Fe ₃ O ₄ magnetic particles were synthesized by hydrothermal method, specifically: dissolving 0.6g FeCl ₃ ·6H ₂ O in 20mL of ethylene glycol, then adding 1.5g of sodium acetate, vigorously stirring for 30min, and then sealing in In an autoclave, heat at 200 °C for 16 h; wash the magnetic particle product several times with ethanol, and dry at 60 °C; dissolve 5 mg each of dichloroethane and N-hydroxysuccinimide in 1 mL of deionized water by vortexing, Prepare the mixed solution, then add 5 mg magnetic particles into the mixed solution, incubate at room temperature for 30 min, collect the magnetic particles with a magnet, wash twice with ultrapure water, and obtain Fe ₃ O ₄ magnetic particles.

Step 2) Specifically: Add biotin secondary antibody with a concentration of 100 μg/mL to 50 mM sodium acetate buffer solution at pH 6.0, then mix with 5 mg of Fe ₃ O ₄ magnetic particles, vortex the mixture, and incubate overnight at 4°C ; wash the mixture twice with PBS solution of pH 7.0, and then incubate at room temperature for 30 min in 50 mM Tris buffer solution of pH 7.2; then wash with PBS solution of pH 7.0 to obtain the biotin secondary antibody nanozyme probe. Finally, the nanozyme probe was dispersed into 1 mL of 5% BSA-PBS solution. Using JEOL 2000FX 200kV transmission electron microscope (TEM) to observe the particle size of the nanozyme probe, the size distribution of MNP shows that the average diameter is 200nm, which can be used for the construction of the nanozyme sensor.

The sample pad, the binding pad immobilized with the biotin secondary antibody nanozyme probe, the nitrocellulose membrane with the detection line and the quality control line, and the absorbent pad are pasted on the bottom plate in sequence to complete the assembly of the test strip.

The present invention also provides a method for detecting Bacillus cereus based on nucleic acid chromatography biosensing technology, comprising the following steps:

S1. Extract the DNA of the sample to be tested, and use the DNA as a template to perform a LAMP-PCR amplification reaction using the above-mentioned LAMP primer set;

S2. Take 10 μL of the LAMP-PCR amplification product of step S1, mix it with 50 μL of reaction buffer, and drop it on the sample pad of the above-mentioned nanozyme nucleic acid chromatography test strip. After 5-20 minutes (preferably, the reaction time is not less than 15 minutes), Add 2 drops of substrate buffer solution to the test line and the quality control line, react for 5 minutes, then observe the color development with the naked eye, and interpret the results: Negative reaction: the quality control line develops color, and the test line does not develop color; Positive reaction: quality control line Both the control line and the test line develop color; failure response: if the quality control line does not develop color, the test fails or the test strip fails.

For quantitative measurement, a portable strip reader combined with strip reader software was used to record the optical intensity of the strip, and the content of Bacillus cereus in the sample to be tested was calculated according to the optical intensity.

Wherein, the reaction buffer in step S2 is: 4×SSC, 0.2m/m% Tween-20.

The substrate buffer used in the present invention is a commercial reagent (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), containing 20×DAB and 20×H ₂ O ₂ .

In the present invention, the LAMP-PCR amplification reaction system is: 1×Bst Thermal Buffer, 0.6M Betaine, 0.5mM dNTPs solution, 1.6μM FIP, 1.6μM BIP, 0.2μM F3, 0.2μM B3, 3.6mM MgSO ₄ , 8U Bst DNA polymerase large fragment, 2 μL template, ddH ₂ O make up to 25 μL.

The LAMP-PCR reaction program was: 65°C for 30min, then 85°C for 3min.

Before step S1, the step of treating the test sample with propidium iodide azide is also included, specifically: add iodine azide at a final concentration of 10 μg/mL to 1 μL of the test sample containing live bacteria or heat-inactivated bacteria Propidium (PMA), incubate in the dark for 5min, then use a 500W halogen light source to expose the sample for 5min, place the sample tube 20cm away from the light source and place it horizontally on ice, shake the sample tube every 30s to keep the light source uniform Exposure; followed by DNA extraction.

The present invention further provides a kit containing the LAMP primer set for detecting Bacillus cereus and/or the nanozyme nucleic acid chromatography test strip.

The present invention has the following advantages:

(1) The present invention firstly develops a nanozyme biosensor combining PMA, LAMP and nanozyme test paper.

(2) Nanozyme test paper was successfully used to distinguish live and dead bacteria cells for the first time.

(3) The nanozyme biosensor of the present invention can be used for on-site diagnostic testing of live Bacillus cereus.

(4) Provide a set of new LAMP primers, which can be used to detect the hbl A gene of Bacillus cereus.

(5) The lower detection limit of the nanozyme sensor for Bacillus cereus can reach 10 CFU/mL.

Description of drawings

Figure 1 shows the results of LAMP amplification of Bacillus cereus in Example 1 of the present invention; wherein, M is DNA Marker, lanes 1-3 are positive amplification of Bacillus cereus, and lane 4 is negative amplification.

Fig. 2 is the optimization to biosensor in the embodiment of the present invention 2; Wherein, (A) the impact of film material on biosensor peak area; (B) the influence of FITC antibody concentration on detection line on biosensor peak area; (C) The effect of the amount and volume of the nanozyme probe on the peak area of the biosensor; (D) the effect of the reaction time on the peak area of the biosensor.

Fig. 3 is a graph showing the performance analysis results of the biosensor in Example 3 of the present invention. Among them, (A) sensor reproducibility analysis result graph; (B) sensor specificity analysis result graph: samples from left to right are: double distilled water, other Enterobacter DNA amplification products, other non-cereus bacillus strains DNA amplification products, DNA amplification products of dead Bacillus cereus strains, DNA amplification products of live Bacillus cereus strains; (C) Sensor stability analysis results: From left to right, the test of the same batch of sensors every other week result.

Fig. 4 is the linear curve that Bacillus cereus detects in the embodiment 4 of the present invention: the variation of peak area with lg (concentration of Bacillus cereus).

Fig. 5 is a structural schematic diagram of the nanozyme nucleic acid chromatography test strip of the present invention. Among them, 1-sample pad, 2-nitrocellulose membrane, 3-binding pad, 4-absorbent pad, 5-test line, 6-quality control line, 7-bottom plate.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Example 1 The method for detecting Bacillus cereus based on nucleic acid chromatography biosensing technology

1. Experimental materials

See Table 1 for information on the strains of Bacillus cereus and non-Bacillus cereus used in this example.

Table 1 Information on Bacillus cereus and Bacillus noncereus used

The specificity of the nanozyme sensor was determined using Bacillus cereus and other bacterial strains. All strains were stored at -80°C in 20% (v/v) glycerol solution until use. They were then cultured overnight in LB medium for activation. Bacillus cereus concentrations were determined spectrophotometrically.

2. Genome extraction of Bacillus cereus

The bacterial genomic DNA extraction kit from New Industry Company was used, and the specific steps were as follows:

(1) Take 1.5 mL of bacterial culture solution, centrifuge at 12,000 g for 1 min, and absorb the supernatant as much as possible.

(2) Add 200 μL of solution A to the bacterial pellet and shake until the bacteria are fully suspended.

(3) Add 20 μL of 10 mg/mL proteinase K to the tube, and mix thoroughly. During this period, the centrifuge tube can be inverted and mixed several times until the sample is completely digested. Clear and viscous liquid is a sign of complete digestion.

(4) Add 2000 μL solution B to the tube and mix well. If a white precipitate appears, it can be placed at 75°C for 15-30 minutes, and the precipitate will disappear without affecting subsequent experiments.

(5) Add 220 μL of absolute ethanol to the tube and mix thoroughly. At this time, flocculent precipitates may appear, which will not affect the extraction of DNA. The solution and flocculent precipitates can be added to the adsorption column and allowed to stand for 2 minutes.

(6) Centrifuge at 12000 g for 1 min, discard the liquid, and put the adsorption column into a collection tube.

(7) Add 700 μL of washing solution to the adsorption column, centrifuge at 12000 g for 1 min, discard the solution, and put the adsorption column into a collection tube.

(8) Add 500 μL of washing solution to the adsorption column, centrifuge at 12000 g for 1 min, discard the solution, and put the adsorption column into a collection tube.

(9) Centrifuge at 12,000 g for 2 minutes, place the adsorption column at room temperature or in a 50°C incubator for several minutes, and remove the residual rinse solution in the adsorption column.

(10) Put the adsorption column into a clean centrifuge tube, add dropwise 500-200 μL of eluent preheated in a 75°C water bath to the center of the adsorption membrane, place at room temperature for 2 minutes, and centrifuge at 12,000 g for 2 minutes.

(11) The eluate obtained by centrifugation was added to the adsorption column, left at room temperature for 2 minutes, and centrifuged at 12000 g for 2 minutes to obtain high-quality bacterial genomic DNA.

3. Propidium iodide azide treatment

Add 1 µL of live or heat-inactivated bacteria to PMA at a final concentration of 10 µg/mL. Incubate samples in the dark for 5 min and mix to allow PMA to penetrate into dead cells. The sample was then exposed for 5 min using a 500W halogen light source. The sample tubes were placed 20 cm away from the light source and placed horizontally on ice to avoid excessive heating. Shake the sample tube every 30 s to maintain uniform exposure to the light source. Genomic DNA of Bacillus cereus was isolated using the Bacterial Genomic DNA Extraction Kit from New Indμstry Company, and the extracted genomic DNA was subjected to LAMP reaction immediately after preparation. In PMA-treated heat-inactivated B. cereus samples, no genomic DNA was extracted and the samples could not be amplified using LAMP. The genomic DNA of dead and live bacteria that were not treated with PMA could be amplified. It can be seen that the amplified bands of dead bacteria were eliminated by PMA treatment, leaving only the amplified bands of live bacteria. This demonstrates that PMA treatment can effectively eliminate the DNA signal of dead bacteria.

4. Primer design

According to the conserved region of the Bacillus cereus gene hbl A published in the literature, the LAMP primer designing software primerexplorer V 4.0 (https://primerexplorer.jp/lamp4.0.0/index. html) Design primers for the gene, including 2 outer primers F3, B3 and 2 inner primers FIP, BIP (Table 2). The 5' end of FIP was labeled with biotin, and the 5' end of BIP was labeled with fluorescein (FITC). Primers were synthesized by Shanghai Invitrogen Biotech Co., Ltd. for subsequent screening optimization.

Primer sequences used in table 2LAMP

5. Amplification of the Bacillus cereus genome

The Bacillus cereus genome was amplified using the loop-mediated isothermal amplification method (Table 3).

Table 3 LAMP reaction system

The LAMP reaction program was as follows: react at 65°C for 30 minutes, and react at 85°C for 3 minutes to inactivate the enzyme. In this experiment, a system of 25 μL per tube was used, in which no Bacillus cereus genome was added as a negative control group. Take 5 μL of the amplified product, mix it with 1 μL of loading buffer, and run electrophoresis on a 2% agarose gel. Typical DNA amplification bands appear in Bacillus cereus (Figure 1), while non-Bacillus cereus strains do not produce any Bands, thus proving that the designed primers have 100% specificity for Bacillus cereus.

6. Preparation of magnetic particles and nanozyme probes

The magnetic particles were synthesized according to the hydrothermal method. Specifically, 0.6g FeCl ₃ ·6H ₂ O was dissolved in 20mL ethylene glycol, then 1.5g sodium acetate was added, stirred vigorously for 30min, then sealed in an autoclave, heated at 200°C for 16h; the magnetic particle product was washed with ethanol for several and dry at 60°C; dissolve 5 mg each of dichloroethane and N-hydroxysuccinimide in 1 mL of deionized water by vortexing to prepare a mixed solution, then add 5 mg of magnetic particles to the mixed solution, and store at room temperature Incubate for 30 min at high temperature, then collect the magnetic particles with a magnet, and wash twice with ultrapure water to obtain Fe ₃ O ₄ magnetic particles.

Add biotin secondary antibody (goat anti-mouse IgG) at a concentration of 100 μg/mL to 50 mM sodium acetate buffer at pH 6.0, then mix with 5 mg of Fe ₃ O ₄ magnetic particles, vortex the mixture, and incubate overnight at 4°C; The mixture was washed twice with PBS solution of pH 7.0, and then incubated in 50 mM Tris buffer solution of pH 7.2 for 30 min at room temperature; then washed with PBS solution of pH 7.0 to obtain the biotin secondary antibody nanozyme probe. Finally, the nanozyme probe was dispersed into 1 mL of 5% BSA-PBS solution. Using JEOL2000FX 200kV transmission electron microscope (TEM) to observe the particle size of the nanozyme probe, the size distribution of MNP shows that the average diameter is 200nm, which can be used for the construction of the nanozyme sensor.

7. Preparation of Nanozyme Sensor

Assembly of nanozyme nucleic acid chromatography test strip (nanozyme test paper): the test strip includes a sample pad, a binding pad, a nitrocellulose membrane and an absorption pad, wherein at least one strip is provided on the nitrocellulose membrane Test line and 1 quality control line.

①Preparation of the sample pad and the binding pad: first paste the double-sided tape on the bottom plate, then paste the binding pad, and then paste the sample pad on the binding pad, the sample pad overlaps with the binding pad by 2-4mm. Wherein the biotin secondary antibody nanozyme probe is immobilized on the binding pad.

②Dilute the FITC antibody and biotin antibody with the optimal buffer solution to the optimal concentration of 1mg/mL respectively. Put the diluted FITC antibody solution into the nozzle 2 of the BIODOT film scriber, and fix it at a position 1.1 cm away from the lower edge of the NC membrane. Put the diluted biotin secondary antibody solution into the nozzle 1 of the BIODOT film scriber, and fix it at At the position of 1.6cm from the lower edge of the membrane. The distance between the detection line (T line) and the quality control line (C line) is 4.5 mm, and sprayed on the T line and C line of the NC membrane at 1.0 μL/cm respectively. The sprayed NC membrane was dried overnight at 37°C for later use. Cut the test paper into 3.8mm wide test paper with a strip cutter, and put the cut test paper into a packaging bag containing a desiccant.

③Paste the above-mentioned sample pad, binding pad, NC film with T-line and C-line, and absorbent pad on the bottom plate in sequence to complete the assembly of the test strip.

8. Detection of Bacillus cereus

After completing the PMA and LAMP operation steps, the nanozyme test paper is used for detection. The detection includes two reaction steps: (1) hybridization reaction; (2) signal enhancement.

Take 10 μL of the LAMP-PCR amplification product, mix it with 50 μL of reaction buffer (4×SSC, 0.2m/m% Tween-20), and drop it onto the sample pad of the above-mentioned nanozyme nucleic acid chromatography test strip, and react for 15 minutes , add 2 drops of substrate buffer solution (commercial reagent containing 20×DAB and 20×H ₂ O ₂ ) on the test line and quality control line, react for 5 minutes, then observe the color development with the naked eye, and interpret the result: negative reaction : The quality control line develops color, but the test line does not develop color; Positive reaction: Both the quality control line and the test line develop color; Failure reaction: If the quality control line does not develop color, the test fails or the test strip is invalid.

For quantitative measurement, use a portable strip reader combined with strip reader software to record the optical intensity of the band, and calculate the content of Bacillus cereus in the sample to be tested based on the optical intensity.

In order to verify the accuracy of the biosensor established in the present invention in the actual sample detection process, the present invention performs standard addition recovery verification on a certain concentration of Bacillus cereus. Infant milk powder purchased from a local supermarket was tested negative for Bacillus cereus by standard culture and colony count methods. Then Bacillus cereus was mixed into the milk powder at the concentration of 10, 10 ³ and 10 ⁵ CFU/mL and mixed well. PMA treatment, genome extraction, LAMP amplification, nucleic acid test strip color development, peak area reading, and quantitative analysis were carried out in sequence. The results showed that the recovery rate of Bacillus cereus was in the range of 101.4±1.2% to 105.2±3.9%. It shows that the sensor detection method established by the present invention can be used for the detection of actual samples (Table 4).

Table 4 Nanoenzyme sensor detects the content of Bacillus cereus in milk powder

The optimization of embodiment 2 nanozyme nucleic acid test strips

The Fe ₃ O ₄ magnetic particles were synthesized according to the hydrothermal method, and then the magnetic particles were incubated with biotin secondary antibody (goat anti-mouse IgG) to prepare nanozyme probes. Use FITC antibody and biotin antibody to draw lines on the T-line and C-line positions on the NC membrane, and assemble them into nanozyme nucleic acid test strips after drying. In order to improve the sensitivity of the nanozyme sensor, it was systematically analyzed by comparing the performance of membrane materials, the concentration of FITC antibody in the detection area, the amount of nanozyme probe, and the reaction time. The results demonstrated that the performance of the nanoenzyme sensor using Millipore 135S nitrocellulose membrane was better (Fig. 2A). The highest signal peak area was obtained with 1 mg/mL FITC antibody and 1 mg/mL goat anti-mouse IgG (Figure 2B). Furthermore, the amount of nanozyme probe affects the hybridization efficiency between the nanozyme probe and the sample, and using 10 µL of nanozyme probe is the optimal volume (Figure 2C). The color reaction time of the test strip is not less than 15 minutes (Figure 2D).

The performance detection of embodiment 3 nano-enzyme sensor

The principle of the nanozyme sensor is as follows. First, the samples were treated with PMA (step 1). PMA can selectively penetrate the damaged cell membrane of dead cells, bind to the DNA in the cell, and make it unavailable for subsequent LAMP amplification, but if it is the intact cell membrane of living cells, PMA cannot enter the cell. Then, many BIO- and FITC-linked duplex DNAs were generated in a short time using LAMP (step 2). In the presence of the specific sequence of the target substance hbl A, it is recognized and amplified by four primers. The third is the visual interpretation of the nanozyme nucleic acid test paper (step 3). The FITC antibody and goat anti-mouse IgG were immobilized on the nitrocellulose membrane by physical adsorption to form the detection area (TL) and quality control area (CL), respectively. If the sample is positive, after LAMP amplification, the 5' end of the target substance is labeled with biotin, the 3' end is labeled with FITC, and the sample solution is combined with the nanozyme probe. The conjugate is then hybridized to the FITC antibody in the detection zone. The formed conjugate continues to migrate along the strip and reacts with the nanozyme probe and goat anti-mouse IgG to develop color in the quality control area. When the DAB/H ₂ O ₂ enzyme substrate is applied to the detection area and the quality control area, the enzymatic reaction between the nanozyme and the DAB/H ₂ O ₂ enzyme substrate will produce a color reaction to enhance the visual effect. In the absence of live Bacillus cereus, only the control zone develops color.

The performance of the nanozyme sensor was evaluated by the following aspects. First, reproducibility is of great importance in evaluating biosensors. The reproducibility of the biosensor was tested by using 100 CFU/mL of live Bacillus cereus (Figure 3A) for a total of five tests. The corresponding RSD value of the optical response is 1.5%, indicating the excellent reproducibility of this nanozyme sensor. Since samples are in practice always a mixture of different bacterial species, we tried to make the detection method more specific. To evaluate the specificity of the nanozyme sensor, double distilled water, DNA of dead Bacillus cereus, DNA of live Bacillus cereus and DNA of other Enterobacter bacterial strains and DNA of other non-Enterobacter bacterial strains were used. The results are shown in Figure 3B, and no false positives were observed. Responses for non-cereus and dead B. cereus remained as low as the background signal, indicating that non-specific adsorption did not significantly affect the reaction system for live B. cereus detection under the experimental conditions. In the early stage, the high specificity of the nanozyme sensor was further improved by using LAMP amplification. For the stability (lifetime) study, the performance of the nanozyme sensor was tested after storage at room temperature for 1-5 weeks. The results were shown in Figure 3C, the response of the nanozyme sensor to 100 CFU/mL Bacillus cereus remained almost the same, indicating that the nanozyme sensor had good stability.

Example 4 Sensitivity Verification of Nanozyme Sensor

In order to evaluate the sensitivity of the nanozyme sensor, sample solutions containing different concentrations of live Bacillus cereus (ranging from 0 to 10 ⁵ CFU/mL) were measured under the optimal experimental conditions, and then the absorption peak area of the TL line was measured, three parallel. In the minimum to maximum range, the resulting graph of the response to the concentration of Bacillus cereus is linear, and the correlation equation is peak area=252781g Bacillus cereus concentration+618.63, and the correlation coefficient R is ^0.9979 , which is suitable for quantitative detection. Wherein, the concentration unit of Bacillus cereus is CFU/mL.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

sequence listing

<110> China Agricultural University

<120> Method for detecting Bacillus cereus based on nucleic acid chromatography biosensing technology

<130> KHP171113507.6

<160> 4

<170> PatentIn version 3.3

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<213> Artificial sequence

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tgctattttg ggtctaccaa 20

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gtggacatat aagtaagagc gtta 24

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acgtaattct gctaataaag gctctaattg gcggtattat agtcgg 46

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aaccttaaat cgtgtagttg gagttatcat caagcgcctt gtg 43

Claims (10)

1. the LAMP primer group for detecting Bacillus cereus (Bacillus cereus), it is characterised in that including：

Outside forward primer F3：5’-TGCTATTTTGGGTCTACCAA-3’；

Outside reverse primer B3：5’-GTGGACATATAAGTAAGAGCGTTA-3’；And

Inner side forward primer FIP：5’-ACGTAATTCTGCTAATAAAGGCTCTAATTGGCGGTATTATAGTCGG-3’；

Inner side reverse primer BIP：5’-AACCTTAAATCGTGTAGTTGGAGTTATCATCAAGCGCCTTGTG-3’；

Wherein, primers F IP 5 ' end mark biotins, primer BIP 5 ' end mark fluorescent element FITC.

2. a kind of nanometer enzymatic nucleic acid chromatograph test strip, it is characterised in that the preparation method of the test strips comprises the following steps：

1)Fe₃O₄The preparation of magnetic particle；

2) preparation of nanometer enzyme probe：By Fe₃O₄Magnetic particle is incubated with biotin secondary antibody, obtains biotin secondary antibody nano enzyme Probe；

3) assembling of nanometer enzymatic nucleic acid chromatograph test strip：The test strips include sample pad, pad, nitrocellulose filter and suction Pad is received, wherein, the nitrocellulose filter is provided with least 1 detection line and 1 nature controlling line；Life is fixed with the pad Thing element secondary antibody nanometer enzyme probe；

1. marking detection line and nature controlling line on nitrocellulose filter with FITC antibody and biotin antibody respectively, dry；

2. above-mentioned sample pad, pad, the nitrocellulose filter with detection line and nature controlling line and absorption pad are pasted successively On bottom plate, the assembling of test strips is completed.

3. test strips according to claim 2, it is characterised in that hydro-thermal method synthesis Fe is utilized in step 1)₃O₄Magnetic particle, Specially：By 0.6-0.8g FeCl₃·6H₂O is dissolved in 20mL ethylene glycol, then adds 1.5-2.0g sodium acetates, stirs 30- 40min, then it is sealed in autoclave, 200 DEG C of heating 16-18h；Magnetic granular product is washed with ethanol, and is dried at 60 DEG C； Dichloroethanes and each 5-8mg of n-hydroxysuccinimide are dissolved in 1mL deionized waters by being vortexed, mixed liquor is made, so 5-8mg magnetic particle is added in mixed liquor afterwards, is incubated 30-40min at room temperature, then magnetic particle is collected with magnet, uses ultra-pure water Washing, produces Fe₃O₄Magnetic particle.

4. test strips according to claim 3, it is characterised in that step 2) is specially：By the μ g/mL of concentration 100 biology Plain secondary antibody is added in 50mM pH 6.0 sodium-acetate buffer, then with 5-8mg Fe₃O₄Magnetic particle mixes, by mixture whirlpool Rotation mixing, 4 DEG C of overnight incubations；Mixture is washed with pH7.0 PBS liquid, then the room in 50mM pH7.2 Tris buffer solutions Temperature is incubated 30-40min；Washed again with pH7.0 PBS liquid, that is, obtain biotin secondary antibody nanometer enzyme probe.

5. test strips according to claim 2, it is characterised in that step 2) and 3) described in biotin secondary antibody be goat-anti Mouse IgG；Nitrocellulose filter described in step 3) is Millipore135S.

6. according to the test strips described in claim any one of 1-5, it is characterised in that detection line is located at fine away from nitric acid in step 3) On the position for tieing up plain film lower edge 1.1cm, nature controlling line is located on the position away from nitrocellulose filter lower edge 1.6cm；Detection line The distance between nature controlling line is 4.5mm, and FITC antibody and biotin antibody are sprayed on into cellulose nitrate respectively by 1.0 μ L/cm In the detection line and nature controlling line of plain film, wherein coated antibody concentration is 0.5-2mg/mL in detection line and nature controlling line.

7. the method based on nucleic acid chromatography biosensor technique detection Bacillus cereus, it is characterised in that comprise the following steps：

S1, extraction testing sample DNA, using DNA as template, LAMP-PCR expansions are carried out using LAMP primer group described in claim 1 Increase reaction；

S2, the LAMP-PCR amplified productions for taking 10 μ L steps S1, claim 2-6 is added drop-wise to after being mixed with 50 μ L reaction buffers In the sample pad of any one test strips, after 5-20min, 2 drop substrate buffer solutions, reaction are added dropwise on p-wire and nature controlling line Colour developing situation, sentence read result are observed by the naked eye after 5min：Negative reaction：Nature controlling line develops the color, and detection line does not develop the color；It is positive anti- Should：Nature controlling line, detection line develop the color；Failure reaction：If nature controlling line does not develop the color, detection failure or test strips failure；

For quantitative measurment, the optics using the portable strip reader record strip band combined with band reader software is strong Degree, the content of Bacillus cereus in testing sample is calculated according to optical strength；

Wherein, reaction buffer is described in step S2：4 × SSC, 0.2m/m% Tween-20；Contain in the substrate buffer solution 20 × DAB and 20 × H₂O₂。

8. according to the method for claim 7, it is characterised in that the system of LAMP-PCR amplified reactions is in step S1：1× Bst Thermal Buffer, 0.6M Betaine, 0.5mM dNTPs solution, 1.6 μM of FIP, 1.6 μM of BIP, 0.2 μM of F3, 0.2 μM of B3,3.6mM MgSO₄, 8U Bst archaeal dna polymerase large fragments, 2 μ L templates, ddH₂O complements to 25 μ L；

LAMP-PCR response procedures are：65 DEG C of 30min, then 85 DEG C of 3min.

9. the method according to claim 7 or 8, it is characterised in that carried out before step S1, in addition to testing sample The step of nitrine propidium iodide processing, it is specially：Final concentration is added in the testing sample for containing viable bacteria or hot inactivation of bacterial to 1 μ L For 10 μ g/mL nitrine propidium iodide, lucifuge is incubated 5min, then using 500W halogen light sources by sample exposure 5min, by sample QC is remotely from light source 20cm and lain in a horizontal plane on ice, shakes sample cell every 30s to keep to the uniform of light source Exposure；Then DNA extractions are carried out.

10. the kit containing any one of LAMP primer group and/or claim the 2-5 test strips described in claim 1.