Paper-based chip sensor for detecting vomiting-type Bacillus cereus based on aptamer isothermal amplification

Abstract

The present invention discloses a paper-based chip sensor for detecting vomiting-type Bacillus cereus based on aptamer constant temperature amplification, and relates to the field of biosensor technology. The paper-based chip sensor includes a dark box and a microfluidic paper-based chip; the dark box includes a box body, a dark box top cover, a camera, an LED light group and a sample slot; the microfluidic paper-based chip includes an identification element, a conversion element and a signal output element. The present invention adopts the above-mentioned paper-based chip sensor, which can complete the detection within 1 hour, has the advantages of visual quantitative detection, portability, and high sensitivity, and provides a research basis for the instant detection of vomiting-type Bacillus cereus. It can solve the current detection method that relies on large-scale instruments and professional technicians, the experiment is complicated, the time is long, the efficiency is low, and there is no efficient immunological detection method for Emetic B.cereus, which cannot meet the needs of rapid and portable detection of Emetic B.cereus in food, especially in rice and flour products.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, in particular to a paper-based chip sensor for detecting vomiting-type Bacillus cereus based on aptamer constant temperature amplification. Background Art

[0002] Bacillus cereus (B.cereus) is a spore-forming, facultative anaerobic Gram-positive bacterium that is commonly found in starchy rice and flour products. B.cereus is a global foodborne pathogen. 5 CFU / g can cause food poisoning and even death. B.cereus can be divided into vomiting type and diarrhea type according to the different gastrointestinal symptoms caused. Vomiting symptoms will occur about 0.5-6 hours after eating food contaminated by Emetic B.cereus. Since the vomitoxin secreted by Emetic B.cereus is resistant to high temperatures and has a low pathogenic dose (less than 10 5 CFU / g), which leads to frequent food poisoning caused by it, accounting for about 75.9% of the total B. cereus food poisoning. Therefore, it is very important to establish an efficient, portable and economical method to detect Emetic B. cereus in food, especially in rice and flour products.

[0003] At present, the plate counting method for detecting Emetic B.cereus has the disadvantages of being time-consuming, low in sensitivity, and large human errors. Molecular biological detection methods rely on large instruments and professional laboratories, which limits the detection capabilities of being easy to carry and easy to operate. In addition, vomitoxin is not immunogenic, and Emetic B.cereus has no related antibodies, which limits the application of immunological methods in Emetic B.cereus.

[0004] In recent years, thanks to its high affinity, high specificity, easy synthesis and modification, nucleic acid aptamers have made significant progress in many research fields including pathogen detection. Cell-SELEX technology is a new method for screening nucleic acid aptamers for pathogenic bacteria, which can be used to screen nucleic acid aptamers from 10 13 ~10 16In addition, pathogenic bacteria have complex outer membrane structures and abundant membrane proteins, which provide many possibilities for the targeted selection of nucleic acid aptamers, making it possible to screen out multiple effective nucleic acid aptamers. Nucleic acid aptamers that specifically recognize target molecules can be used as molecular recognition elements (MREs) on biosensors. After the interaction between nucleic acid aptamers and target molecules, chemical, electrical or optical signal changes are generated, thereby detecting targets in samples.

[0005] Microfluidic paper-based analytical devices (μPADs) use filter paper as the substrate, and use various processing techniques to form a hydrophilic / hydrophobic microchannel network. Micro sensors are built on paper to achieve a series of experimental operations in a micro range using micro devices. μPADs are widely used in medical diagnosis, food safety testing, environmental monitoring and other fields because of their simplicity, low cost and portability. Summary of the invention

[0006] The purpose of the present invention is to provide a paper-based chip sensor for detecting vomiting type Bacillus cereus based on aptamer constant temperature amplification, which can complete the detection within 1 hour, has the advantages of visual quantitative detection, portability, high sensitivity, etc., and provides a research basis for the instant detection of vomiting type Bacillus cereus. It can solve the current detection method that relies on large-scale instruments and professional technicians, the experiment is complicated, the time is long, the efficiency is low, and there is no efficient immunological detection method for Emetic B.cereus, which cannot meet the rapid and portable detection needs of Emetic B.cereus in food. At the same time, the aptamer of vomiting type Bacillus cereus is obtained by Cell-SELEX technology screening, which has high affinity and specificity.

[0007] To achieve the above-mentioned object, the present invention provides a paper-based chip sensor for detecting vomiting-type Bacillus cereus based on aptamer constant temperature amplification, comprising a dark box and a microfluidic paper-based chip;

[0008] The dark box includes a box body, a dark box top cover, a camera, an LED light group, a sample slot and a small power bank;

[0009] The microfluidic paper-based chip includes a recognition element, a conversion element and a signal output element;

[0010] The recognition element refers to a nucleic acid aptamer of Bacillus cereus immobilized on a paper-based chip through the interaction of biotin and streptavidin, wherein the Bacillus cereus aptamer is obtained by screening through the Cell-SELEX technology;

[0011] The aptamers were obtained by screening using the Cel1-SELEX technology with Emetic B.cereus as positive screening bacteria and E.coli K12, S.aureus, B.thuringiensis, and B.cereus as counter-screening bacteria. The nucleic acid aptamer sequences are shown as DL-1, DL-2, and DL-3.

[0012] The screening process is as follows:

[0013] (1) The initial random ssDNA library (DL) designed by the present invention for screening is composed of a 76nt random sequence, and its 5' and 3' ends each contain an 18nt fixed sequence as a primer binding site, and a 40nt random region sequence is inserted between the two as the core site for target binding. Carboxyfluorescein-modified upstream primer (FAM-FP) and biotin-modified downstream primer (Biotin-RP) are mainly used to prepare the ssDNA library required for the next round of screening and monitor the enrichment of the library. The specific nucleic acid sequence is shown in the following table. The initial random ssDNA library and primers (dry powder) must be centrifuged (10000rpm, 10min) and then precipitated with ddH 2 Dissolve in 5% dHO into a 100 μM stock solution and store at -20°C. Before use, take an appropriate amount of the stock solution to prepare a 10 μM DNA stock solution.

[0014]

[0015]

[0016] Note: N40 is a 40nt random sequence, and the probability of A, T, C and G appearing at each position is equal.

[0017] (2) Dissolve the initial random ssDNA library in BB, heat (95°C, 6 min) to denature, and immediately transfer to ice (4°C, 10 min). Mix the denatured ssDNA library with Emetic B.cereus bacteria in a volume of 1 mL and incubate on a shaker (room temperature, 200 rpm). After incubation, transfer the ssDNA library and bacteria mixture to a new centrifuge tube, centrifuge (4°C, 6,000 rpm, 3 min), remove the supernatant, add 500 μL EB, resuspend by pipetting, centrifuge (4°C, 6,000 rpm, 3 min), and wash 3 to 5 times. After washing, resuspend the bacteria in 100 μL ddH 2 O, heat and disrupt the bacteria (100°C, 10 min) to make the firmly bound ssDNA fall off from the E. cereus bacteria, cool naturally to room temperature, centrifuge (12,000 rpm, 10 min), take the supernatant, and resuspend the bacteria in 100 μL ddH 2O for two times to obtain 300 μL of ssDNA library supernatant.

[0018] (3) Using the ssDNA library collected in each round of screening as a template, add FAM-FP and Biotin-RP to perform PCR amplification to obtain a sufficient amount of dsDNA library. The specific reaction system of batch PCR is shown in the table below. After the amplification is completed, the PCR amplification product is analyzed by 3% agarose gel electrophoresis.

[0019]

[0020] (4) 100 μL of streptavidin-agarose beads (SA-agarose beads) were centrifuged (5000 rpm, 2 min) to remove the ethanol solution protecting the beads, and 500 μL of PBS was added for resuspending by pipetting, and then centrifuged (5000 rpm, 2 min) to wash twice.

[0021] (5) 1 mL of batch PCR amplification product was mixed with SA-agarose microbeads and incubated (25°C, 30 min). SA and Biotin interacted to fix dsDNA on agarose microbeads. The supernatant was removed by centrifugation (5000 rpm, 2 min), and then resuspended by pipetting with 500 μL PBS and washed twice by centrifugation (5000 rpm, 2 min). 200 μL of NaOH solution (200 mM) was added for reaction (25°C, 15 min) to dissociate the DNA double strands, followed by centrifugation (5000 rpm, 2 min), and the supernatant was collected. 150 μL of NaOH solution was added, and the mixture was resuspended by pipetting. The mixture was washed twice by centrifugation (5000 rpm, 2 min), and the supernatant was collected to obtain 500 μL of ssDNA product.

[0022] (6) Add 500 μL of ssDNA product to the balanced desalting column. Do not collect the droplets at this time. Wait until they are completely dripped and add 1 mL of ddH 2 O was used to elute and the dripping solution was collected. The collected aqueous solution was detected by ultraviolet spectrophotometer and the characteristic absorption value (OD 260 ), the DNA content was calculated by the following formula; finally, the aqueous solution was freeze-dried into powder and stored at -20°C for future use.

[0023] n=(OD 260 ×33μg) / (76×330g / mol)

[0024] Of which: 1OD 260 The mass of DNA is about 33 μg, the average relative molecular mass of each base is about 330 g / mol, and n is the molar amount of DNA contained in 1 mL of aqueous solution.

[0025] (7) The ssDNA library was first incubated with the reverse screening bacteria, centrifuged (4°C, 6000 rpm, 3 min) to remove the ssDNA bound to the reverse screening bacteria, and the supernatant was taken and incubated with Emetic B. cereus.

[0026] (8) After the screening is completed, the ssDNA library obtained in the final round is amplified by PCR, and the PCR amplification products are sequenced by high-throughput sequencing. After the samples are sequenced, DNAMAN 6.0 analysis software is used to compare the homology of the sequences and draw a family evolutionary tree. Based on the analysis results, the best nucleic acid aptamer sequences DL-1, DL-2, and DL-3 are selected.

[0027] The transformation element converts the EmeticB.cereus signal into a pH signal generated by urease accumulation through a constructed super sandwich hybridization probe, and converts a 1:1 recognition signal into a 1:N colorimetric signal output; the transformation element is a super sandwich hybridization probe, and the composition sequence is shown as DL-1-YC, UM-DNA, and DL-4.

[0028] The signal conversion element means that when Emetic B.cereus is present, Emetic B.cereus simultaneously binds to DL-2 and DL-1-YC fixed on μPAD, and a super sandwich hybridization trigger chain DL-4 and urease-DNA (UM-DNA) are added. DL-1-YC first complementarily hybridizes with UM-DNA, and UM-DNA is continuously connected through DL-4 to obtain DL-1 (N-UM-DL-1) containing N ureases.

[0029] The signal output element is used to photograph the sample slot through a camera in the dark box for imaging processing, obtain photos and perform RGB value analysis after connecting to a smart phone through a data cable, and obtain the concentration of the target bacteria in combination with the standard curve.

[0030] The paper-based chip consists of a hydrophilic circular sample-adding area and a hydrophobic non-sample-adding area.

[0031] The method for making the paper-based chip is to design the pattern of the microfluidic paper-based chip with the help of AutoCAD 2020 software. The pattern is printed on the surface of Whatman No. 1 filter paper using a Xerox paraffin printer, and the filter paper is placed in a 90°C oven and heated for 45 seconds. After the printed wax pattern melts and penetrates into the paper substrate to form a hydrophobic channel, it is cooled at room temperature for use. DL-2 is fixed on the surface of the microfluidic paper-based chip through the action of biotin and streptavidin.

[0032] The present invention also provides the use of the paper-based chip sensor in the detection of vomiting-type Bacillus cereus, and the application is not for the purpose of disease diagnosis.

[0033] The construction method of the above-mentioned paper-based chip sensor is as follows:

[0034] (1) 1 nmol DL-1-NH and 3.2 μL MBS (3-maleimidobenzoic acid-N-succinimidyl ester) solution were mixed respectively, and the volume was made up to 100 μL with 1× PBS buffer. The mixture was reacted at room temperature for 2 h, and then centrifuged in a 3000 Dalton ultrafiltration centrifuge tube (12,000 rpm, 10 min). 100 μL 1× PBS buffer was added, and the mixture was resuspended by pipetting. The mixture was washed three times by centrifugation (12,000 rpm, 10 min) to remove excess MBS. The liquid retained in the ultrafiltration centrifuge tube was MBS-DNA, which was collected and resuspended in 100 μL 1× PBS buffer. The concentration was about 10 μM.

[0035] (2) 1 mL of urease solution and MBS-DNA were mixed and reacted at room temperature in the dark for 2 h to allow the urease and DNA to couple. The mixture was then centrifuged in a 100K Dalton ultrafiltration centrifuge tube (9,000 rpm, 5 min). 50 μL of 1× PBS buffer was added and resuspended by pipetting. The mixture was washed three times by centrifugation (9,000 rpm, 5 min). The liquid retained in the ultrafiltration centrifuge tube was the urease-DNA conjugate (UM-DNA). The UM-DNA was collected and resuspended in 100 μL of 1× PBS buffer. The concentration of UM-DNA was about 10 μM and stored at 4°C for later use.

[0036] (3) A circle with a radius of 6 mm was designed using Auto CAD 2020 software. The hydrophobic pattern was printed on the filter paper using a wax printer. The filter paper was heated at 85 °C for 5 min to melt the paraffin and penetrate into the filter paper to form a hydrophobic paraffin barrier. The filter paper was then cooled at room temperature for later use.

[0037] (4) Soak the paper chip in glutaraldehyde solution for 30 minutes, then take it out and wash it with deionized water for 15 minutes to remove the non-specific binding part. After washing, place it in an oven to dry, then take it out and add 10 μL of 50 μg / mL streptavidin solution to the surface of the glutaraldehyde-modified paper chip, incubate it at room temperature for 10 minutes, dry it at room temperature to form a streptavidin-modified paper chip, and store it in a refrigerator at 4°C.

[0038] (5) Add 10 μL of biotinylated aptamer DL-2 (10 μM) to the streptavidin paper chip and incubate at room temperature for 10 min. 2 O for 15 min to remove excess biotinylated aptamers and store at 4°C for later use.

[0039] The present invention also provides a detection method for the above-mentioned microfluidic paper-based chip sensor, comprising the following steps:

[0040] Step 1: Add the sample solution and N-UM-DL-1 droplets to the circular area on the microfluidic paper chip and wait for the reaction for 25 minutes;

[0041] Step 2: Use ddH 2 O cleaning for 5 minutes;

[0042] Step 3: Add urea phenol red indicator at pH ~ 5 and react for 30 minutes to observe the color change of the paper-based chip;

[0043] Step 4: Place the microfluidic paper-based chip into the sample slot of the dark box, connect the smartphone to the data cable of the dark box, obtain the image and RGB value information, and determine the concentration of Emetic B.cereus based on the standard curve of R / G value.

[0044] SI: Preparation of standard curve: Prepare samples of Emetic B.cereus bacterial solution with different concentrations, and the detection steps are the same as above. Use the color processing software Color Picker to perform RGB analysis on its color, and take the R / G value for quantitative analysis. Use the logarithm of the Emetic B.cereus bacterial solution concentration as the horizontal axis and the R / G value in the sample fluorescence signal as the vertical axis to draw a standard curve;

[0045] SII: Detect the sample according to the above detection method, substitute the R / G value in the fluorescence signal of the sample to be tested into the standard curve, calculate the content of Emetic B.cereus in the sample, and realize the quantitative detection of Emetic B.cereus;

[0046] SIII: Emetic B.cereus concentration at 10 1 ~10 6 There is a good linear relationship between CFU / mL and R / G value, and the relationship obtained is R / G=0.331x+0.8167, R 2 =0.986.

[0047] The present invention also provides application of the above detection method in detecting vomiting-type Bacillus cereus in food.

[0048] Furthermore, the food is a rice and flour product with a high starch content, including rice and rice noodles.

[0049] The detection and analysis principle of the paper-based chip sensor in the present invention is:

[0050] (1) Urease-DNA (UM-DNA) first hybridizes with DL-1-YC, then with the 5' of DL-4, and the 3' of DL-4 hybridizes with the new UM-DNA. The hybridization is repeated continuously to obtain DL-1 containing N ureases (N-UM-DL-1);

[0051] (2) DL-2 was fixed on the μPAD surface through the action of Biotin and SA. Then N-UM-DL-1 and the sample to be tested containing Emetic B.cereus were added to the microfluidic paper-based chip (μPAD) for incubation. Emetic B.cereus was captured by DL-2 on the μPAD. At the same time, N-UM-DL-1 also combined with Emetic B.cereus to form a DL-2-Emetic B.cereus-UM sandwich structure. The unfixed N-UM-DL-1 and Emetic B.cereus were washed and removed. Urea phenol red indicator (pH ~ 5) was added. Urease hydrolyzed urea, causing the pH of the reaction system to increase, and the μPAD changed from bright yellow to rose red.

[0052] (3) The microfluidic paper-based chip is placed in the sample slot of the dark box. The camera of the dark box will take pictures and display them on the mobile phone through the data cable to obtain the RGB value of its fluorescence signal. The concentration of Emetic B. cereus can be determined by analyzing the RGB and combining it with the standard curve.

[0053] The advantages and positive effects of the paper-based chip sensor for detecting vomiting-type Bacillus cereus based on aptamer constant temperature amplification described in the present invention are:

[0054] 1. The present invention uses nucleic acid aptamers DL-1 and DL-2 as recognition probes, uses super sandwich hybridization for signal amplification, and combines microfluidic paper-based chips for signal reporting to construct a visual quantitative biosensor. The sensor can specifically detect Emetic B.cereus, 10 2 ~10 8 CFU / mL Emetic B.cereus can be observed to change color, with a lower detection range, Emetic B.cereus concentration and R / G value have a good linear relationship, the linear response range is 10 1 ~10 6 CFU / mL. In the rice system, the sensor also has good detection ability.

[0055] 2. The paper-based chip sensor in the present invention can complete detection within 1 hour, and has the advantages of visualized quantitative detection, portability, and high sensitivity. It is expected to become an effective tool for detecting Emetic B. cereus in food, especially high-starch rice and flour products.

[0056] 3. The present invention screened the aptamer of Emetic B.cereus and designed a microfluidic paper-based chip sensor using the aptamer as the recognition element for detection, which provides a certain theoretical basis for the detection of bacteria and the diagnosis of related diseases in the future. The designed microfluidic paper-based chip sensor is expected to combine with the aptamer of other foodborne pathogens, so that it can be used for real-time and multiple detection of pathogens in food matrices.

[0057] 4. The present invention constructs a paper-based chip sensor that does not rely on an external mobile phone to take pictures, and can obtain colorimetric signals using a built-in camera.

[0058] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a schematic diagram of the composition structure of the paper-based chip sensor in the present invention;

[0060] Figure 2 This is a schematic diagram of the principle of screening for Emetic B.cereus aptamers in the present invention;

[0061] Figure 3 The library enrichment status of the 2nd to 12th round of screening process in the present invention;

[0062] Figure 4 For monitoring the screening process in the present invention, A is the binding rate analysis of each round of screening; B is the fluorescence image of the binding of FAM-ssDNA and Emetic B.cereus in rounds 7 to 12;

[0063] Figure 5 It is a homology comparison diagram of the sequences in the present invention;

[0064] Figure 6 is the family evolutionary tree of the sequences in the present invention;

[0065] Figure 7 Schematic diagram of the secondary structure of nucleic acid aptamers DL-1, DL-2 and DL-3 in the present invention, from left to right are DL-1, DL-2 and DL-3;

[0066] Figure 8 To investigate the binding ability of nucleic acid aptamers DL-1, DL-2, and DL-3 with Emetic B.cereus in the present invention;

[0067] Fig. 9 The figure is a graph showing the Kd values ​​of DL-1, DL-2, and DL-3 in the present invention, wherein A is the Kd value result of DL-1, B is the Kd value result of DL-2, and C is the Kd value result of DL-3;

[0068] Fig.10 A is the 3% agarose gel electrophoresis analysis of urease-DNA conjugates of the present invention, 1-DL-NH, 2-Urease+DL-NH, 3-Urease+MBS+DL-NH (UM-DNA); B is the UV spectrum of UM-DNA activity, and the inset is the corresponding photograph of the solution;

[0069] Fig.11 10% non-denaturing polyacrylamide gel electrophoresis is used to verify the formation of the probe in the present invention;

[0070] Fig.12 The paper-based chip of the present invention is fabricated, wherein A is a physical image of the microfluidic paper-based chip, B is a scanning electron microscope image of the filter paper that has not been processed by wax printing, and C is a scanning electron microscope image of the filter paper that has formed a paraffin barrier;

[0071] Fig.13 The feasibility of the paper-based chip in the present invention for detecting Emetic B.cereus;

[0072] Fig.14 This is a selectivity verification diagram for the paper-based chip detection in the present invention;

[0073] Fig.15 This is a sensitivity test diagram of the paper-based chip detection in the present invention;

[0074] Fig.16 A is a relationship diagram of the R / G values ​​detected by different concentrations of Emetic B.cereus and UM-DL-1 / N-UM-DL-1 of the present invention; B is a linear relationship diagram between the concentration of Emetic B.cereus and the R / B value;

[0075] Fig.17 This is a comparison of the plate count method and the microfluidic paper-based sensor in the present invention for detecting Emetic B.cereus in a high starch system (ns: no significant difference, ****p<0.0001).

[0076] Reference numerals

[0077] 1. Dark box top cover; 2. Camera; 3. LED light set; 4. Sample slot; 5. Power bank; 6. Box body. DETAILED DESCRIPTION

[0078] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0079] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0080] Example 1 Composition of microfluidic paper-based chip sensor

[0081] Microfluidic paper chip sensor composition Figure 1 As shown, it includes a dark box for observing fluorescence and a microfluidic paper-based chip. The dark box includes a box body 6, a dark box top cover 1, a camera 2 for taking pictures and imaging, an LED light group 3 for providing a standard visible light source, a sample slot 4 for carrying the microfluidic paper-based chip, and a small power bank 5 for powering the LED light and the camera.

[0082] When in use, the sample liquid and N-UM-DL-1 liquid are added to the microfluidic paper-based chip, and the paper-based chip is placed in the sample slot after the reaction. The camera takes a picture and transmits the image to the mobile phone through the data cable, and the RGB value can be analyzed. Combined with the standard curve, the concentration of Emetic B.cereus can be quantified.

[0083] Example 2 Screening of recognition elements in microfluidic paper-based chip sensors

[0084] Cell-SELEX technology was used to screen Emetic B.cereus nucleic acid aptamers as recognition elements of the microfluidic paper-based chip proposed in the present invention.

[0085] The specific screening process is:

[0086] In order to obtain nucleic acid aptamers that specifically bind to Emetic B.cereus, Emetic B.cereus was used as positive screening bacteria, and E. coli K12, S. aureus, B. thuringiensis, and B. cereus were used as counter-screening bacteria, and screening was performed using the Cel1-SELEX technology. The screening process is as follows: Figure 2As shown in the figure, Emetic B.cereus was first incubated with the ssDNA library for positive screening, and the ssDNA bound to Emetic B.cereus was retained, and the unbound ssDNA was removed. Then, the collected ssDNA library was used as a template, FAM-FP and Biotin-RP were added for PCR amplification, and SA-agarose beads were added to capture the dsDNA of the PCR amplification product. The ssDNA was prepared by alkaline denaturation, and freeze-dried before being put into the next round of screening. In order to obtain high-affinity and high-specificity nucleic acid aptamers, counter-screening was introduced in the fifth round, that is, the ssDNA library was first incubated with counter-screening bacteria. The counter-screening bacteria strains and incubation time are detailed in Table 1. The ssDNA bound to the counter-screening bacteria was removed, and the unbound ssDNA was subjected to positive screening with Emetic B.cereus. During the screening process, the enrichment of the library was monitored by binding rate and binding force. The greater the binding rate, the more library is bound to the bacteria. When the binding force between the screening library and Emetic B.cereus no longer increases, it indicates that the binding between the library and Emetic B.cereus has reached saturation. The screening can be terminated and PCR amplification of this round of library can be performed for high-throughput sequencing.

[0087] Table 1 Pressure of each round of SELEX screening

[0088]

[0089] During the Cell-SELEX screening process, FAM-FP and Biotin-RP were used to perform PCR amplification and enrichment of the ssDNA library to obtain sufficient dsDNA. Biotin in dsDNA binds to SA-agarose microbeads and denatures under the action of alkali to obtain ssDNA containing FAM (FAM-ssDNA), which is used to monitor the enrichment of the ssDNA library. In each round of SELEX, the changes in fluorescence values ​​before and after incubation of the FAM-ssDNA library with Emetic B.cereus were examined by a fluorescence spectrophotometer, such as Figure 3 As shown, the binding rate of FAM-ssDNA to Emetic B.cereus was calculated.

[0090] The higher the binding rate, the more FAM-ssDNA is bound to the bacteria, which reveals the high affinity between the ssDNA sequence and EmeticB.cereus. Figure 4As shown in A, in the process of gradually increasing the screening pressure, a trend of first decreasing and then increasing binding was observed. In the 5th and 6th rounds of screening, E. coli K12 and S. aureus of different genera were introduced as counter-screening strains, and the sequences that could bind to these strains were eliminated, resulting in a significant decrease in the binding rate. In the subsequent 7th and 8th rounds, when B. thuringiensis and B. cereus of the same genera were added as counter-screening strains, the sequences that specifically bound to Emetic B. cereus began to gradually enrich, causing the binding rate to show an upward trend. Starting from the 9th round, with the continuous increase in screening pressure, the binding ability of the ssDNA library to Emetic B. cereus continued to increase, and the binding rate reached 14% in the 12th round. Further monitoring of the fluorescence values ​​of the FAM-ssDNA prepared in the 7th to 12th rounds after incubation with Emetic B. cereus revealed that there was no significant change in the fluorescence values ​​from the 10th to 12th rounds ( Figure 4 B), which indicates that the binding of FAM-ssDNA to Emetic B.cereus is close to saturation. Therefore, the SELEX screening process was terminated after the completion of the 12th round. Finally, the ssDNA obtained in the 12th round was amplified by PCR, and then the amplified product was subjected to high-throughput sequencing. The results of high-throughput sequencing are shown in Table 2. The sequences with the top 20 repetition rates were selected and named DL-1'~DL-20' (wherein the sequences of DL-1'~DL-20' are shown in SEQ ID NO.7-26), and then the top 20 sequences were subjected to homology analysis using DANMAN 6.0 software ( Figure 5 As shown) and draw the family evolutionary tree ( Figure 6 The repetition rate of DL-1 in the first 20 sequences was higher than that of other sequences. The similarity of the 20 sequences selected in the homology analysis was 50.23%, and they were divided into 4 major families.

[0091] Table 2 Random region sequences of sequencing results

[0092]

[0093]

[0094] Combining homology comparison with family evolutionary tree analysis, the sequences with the highest repetition rate, DL-1', DL-2' and DL-3', were selected as representative sequences in the four major families. An 18nt upstream primer was added to the 5' of these three sequences, and an 18nt downstream primer was added to the 3' to obtain aptamers DL-1, DL-2 and DL-3 with a total length of 76nt. Mfold was then used to predict the secondary structure of the three aptamers. The results are as follows Figure 7 As shown, the three nucleic acid aptamers are mainly composed of stem-loop structures.

[0095] In order to investigate the binding of the three selected aptamers to Emetic B.cereus, we used flow cytometry to investigate the binding ability of the three aptamers to Emetic B.cereus. The flow cytometry results are shown in Figure 2. Figure 8 As shown, the binding ability of RandomLibrary to Emetic B.cereus is lower than that of the three aptamers, indicating that DL-1, DL-2 and DL-3 have better binding ability to Emetic B.cereus.

[0096] The affinity of the three aptamers to Emetic B.cereus was further investigated by measuring the Kb of the aptamers. The concentration of the aptamers was used as the horizontal axis, and the geometric mean of the fluorescence obtained after the aptamers of different concentrations combined with Emetic B.cereus was used as the vertical axis. The Kd of the aptamers was obtained by curve fitting calculation (GraphPad Prism 9 data processing software) using the single-point adsorption equation Y=Bmax X / (Kd+X). Fig. 9 As shown in the figure, the dissociation constants of DL-1, DL-2 and DL-3 were calculated to be 27.37±6.6nM, 49.38±7nM, and 88.65±0.79nM, respectively, all at the nanomolar level, indicating that the nucleic acid aptamers obtained by Cell-SELEX technology screening have good affinity with Emetic B.cereus, among which DL-1 has the strongest affinity. Therefore, DL-1 and DL-2 were selected as the recognition elements in the microfluidic paper-based chip sensor.

[0097] Example 3 Construction of microfluidic paper-based chip based on nucleic acid aptamers

[0098] The present invention uses nucleic acid aptamers DL-1 and DL-2 as recognition probes, combines them with microfluidic paper-based chips for signal reporting, and uses super sandwich hybridization for signal amplification to construct a biosensor for visual quantitative detection of Emetic B. cereus.

[0099] Sandwich hybridization is used for signal amplification. The designed DL-1-YC and UM-DNA are continuously extended by complementary hybridization through the super sandwich hybridization trigger chain DL-4 to obtain DL-1 (N-UM-DL-1) containing N ureases, thereby improving the sensitivity of detecting Emetic B.cereus. DL-NH reacts with MBS to generate maleimidobenzoic acid DNA amide (MDA), and then urease is coupled to the double bond of malondialdehyde in maleimide through hydrogen sulfide bond addition. Fig.10In Figure A, it can be observed that when DL-NH has not reacted with MBS to generate MDA, urease cannot be directly coupled with DL-NH. Urease can only be successfully coupled to DL-NH after DL-NH has reacted with MBS to generate MDA.

[0100] UM-DNA and ssDNA were added to a solution containing urea phenol red indicator (pH ~ 5) respectively. After the reaction, the color change was observed and the absorbance was measured using an ultraviolet spectrophotometer. Fig.10 As shown in Figure B, in the presence of urease, urea is hydrolyzed to produce ammonia, which leads to an increase in the pH value of the reaction system, resulting in a significant change in the color of the reaction system from bright yellow to rose red. In addition, the absorption peak is red-shifted and a peak appears at 560nm. This shows that urease still maintains enzyme activity after being successfully coupled to DL-NH through MBS. After DL-NH was coupled to urease (UM-DNA) through MBS, appropriate amounts of DL-4 and DL-1-YC were added to Mg 2+ Under the catalytic action of , a nucleic acid aptamer (N-UM-DL-1) connected to N ureases was obtained. The results were verified by 10% non-denaturing polyacrylamide gel electrophoresis. Fig.11 As shown, DL-4 can base pair with DL-NH to form a long chain. DL-1-YC can bind to DL-NH and form a long chain of DL-4+DL-NH+DL-1-YC through chain hybridization switching of DL-4. The sequences used are shown in Table 3.

[0101] Table 3 Nucleotide sequences

[0102]

[0103] Note: F is carboxyfluorescein (FAM), B is biotin, and NH is amino. Sequences with a single underline "" can base pair with each other, and sequences with a double underline "" can base pair with each other. sequences can base pair with each other.

[0104] The Whitman filter paper is printed with wax to generate the corresponding pattern, and then heated at high temperature. The paraffin melts and can penetrate and cover the surface of the filter paper, forming a paraffin barrier with hydrophobic properties. The filter paper that has not been printed with wax and the filter paper that has formed a paraffin barrier are imaged and characterized by scanning electron microscopy. Fig.12 In the middle A, the filter paper without wax printing treatment can be observed with abundant pores such as Fig.12 In B, after the wax printing, the pores on the filter paper surface are almost blocked by paraffin after heating, forming a hydrophobic barrier. Fig.12 Middle C.

[0105] Example 4: Method for using microfluidic paper chip sensor

[0106] (1) Add sample solution and N-UM-DL-1 droplets to the circular area on the microfluidic paper chip and allow to react for 25 minutes.

[0107] (2) Use ddH 2 O for 5 minutes to wash away unfixed bacteria, N-UM-DL-1, etc.

[0108] (3) Add urea phenol red indicator (pH ~ 5) and react for 30 minutes, and observe the color change of the paper-based chip.

[0109] (4) Place the microfluidic paper-based chip into the sample slot of the dark box, connect the smart phone to the data cable of the dark box, obtain the image and RGB value information, and determine the concentration of Emetic B. cereus according to the standard curve of R / G value.

[0110] Example 5 Feasibility of microfluidic paper-based chip for detecting Emetic B. cereus

[0111] Emetic B.cereus+N-UM-DL-1, B.cereus+N-UM-DL-1, B.thuringiensis+N-UM-DL-1, S.aureus+N-UM-DL-1, and E.coli K12+N-UM-DL-1 were reacted on the paper substrate for 30 min, and then washed with ddH 2 O for 15 min to wash away the unfixed bacteria and N-UM-DL-1, then add urea phenol red indicator (pH ~ 5) and react for 30 min, observe the color change of the paper chip and take photos for record. Fig.13 As shown in Figure A, outside the paper substrate, after incubation of Emetic B.cereus with UM-DL-1 and washing with BB three times, urea phenol red indicator (pH ~ 5) was added, and a change from bright yellow to rose red was observed. A change from bright yellow to rose red was also observed on the paper substrate, indicating that UM-DL-1 can bind to Emetic B.cereus and maintain the activity of the enzyme. Fig.13 As shown in B, only the paper-based chips with immobilized nucleic acid aptamer DL-2, EmeticB.cereus, and UM-DL-1 / N-UM-DL-1 can show color changes, and the higher the content of urease, the more obvious the color change.

[0112] Example 6 Specificity of microfluidic paper-based chip for detecting Emetic B. cereus

[0113] Emetic B.cereus+N-UM-DL-1, B.cereus+N-UM-DL-1, B.thuringiensis+N-UM-DL-1, S.aureus+N-UM-DL-1, and E.coli K12+N-UM-DL-1 were added to the paper-based chip for reaction for 30 min and then washed with ddH 2 O for 5 minutes, add urea phenol red indicator (pH ~ 5), and react for 30 minutes. Fig.14 As shown in A, the color of the microfluidic paper-based chip with B.thuringiensis, S.aureus, B.cereus, and E.coli K12 added did not change significantly. However, the microfluidic paper-based chip with Emetic B.cereus added showed the generation of rose red, indicating that the paper-based sensor has good specificity. At the same time, the color processing software Color Picker was used to perform RGB analysis on its color, and the R / G value was taken for qualitative analysis, as shown in Figure 1. Fig.14 As shown in B, the R / G value of Emetic B.cereus is significantly higher than that of other bacterial strains, indicating that this detection method also has good selectivity for Emetic B.cereus.

[0114] Example 7 Sensitivity of microfluidic paper-based chip in detecting Emetic B. cereus

[0115] Different concentrations of Emetic B.cereus and UM-DL-1 / N-UM-DL-1 were reacted on the paper-based chip for 30 min, and then ddH 2 O for 5 minutes, add urea phenol red indicator (pH ~ 5), and react for 30 minutes. Fig.15 As shown, when UM-DL-1 is added, the content of 10 4 The paper-based chip of CFU / mL Emetic B.cereus showed obvious color change, and after adding N-UM-DL-1 to increase the content of urease, 10 2 CFU / mLEmetic B.cereus can be observed with obvious color changes.

[0116] Use the color processing software Color Picker to perform RGB analysis on its color and take the R / G value for quantitative analysis. Fig.16 As shown in A, the R / G value showed an upward trend with the increase of Emetic B.cereus concentration, and the R / G value of N-UM-DL-1 was higher than that of UM-DL-1; Fig.16 As shown in B, when UM-DL-1 was added, the concentration of Emetic B.cereus was 10 2 ~108 CFU / mL and R / G value have a good linear relationship, and the relationship obtained is R / G=0.3425x+0.4410, R 2 =0.9825; when N-UM-DL-1 was added, the concentration of Emetic B.cereus was 10 1 ~10 6 There is a good linear relationship between CFU / mL and R / B value, and the relationship obtained is R / G=0.331x+0.8167, R 2 =0.986. When the urease content increased, the paper-based sensor had a lower detection range, indicating that the construction of a portable biosensor based on nucleic acid aptamers has good detection performance, and the color of the paper-based chip can be recognized by a smartphone to achieve visual quantitative detection of Emetic B.cereus.

[0117] Example 8 Microfluidic paper chip sensor for rice sample detection

[0118] The ability of microfluidic paper-based chips and plate counting methods to detect Emetic B.cereus in a rice system was compared. Emetic B.cereus was inoculated into the rice system for culture, and the cultured bacterial solution was taken for gradient dilution plate counting. A series of dilution gradients were set up, and each dilution solution was inoculated onto a plate. Three sets of parallel samples were made, and plates with colony counts between 30 and 300 CFU were selected for counting to obtain the total number of colonies on the plate. At the same time, the bacterial solution was detected using a microfluidic paper-based chip, and its color was quantitatively analyzed using RGB using the color processing software Color Picker. Fig.17 As shown in the figure, the plate count method and the microfluidic paper chip obtained consistent results for the four groups of bacterial liquids, indicating that the microfluidic paper chip has good accuracy in the rice system. The microfluidic paper chip is superior to the plate count method in terms of detection time (<1h), solution volume required for detection (<100μL), and detection cost, and the detection accuracy is equivalent to the plate count method.

[0119] The present invention focuses on the screening of nucleic acid aptamers for Emetic B.cereus and the construction of portable detection of Emetic B.cereus based on nucleic acid aptamers. Emetic B.cereus is used as the target bacterium for the screening of nucleic acid aptamers, and nucleic acid aptamers are obtained as molecular recognition elements. Combined with super sandwich hybridization and microfluidic paper-based chip technology, a rapid, sensitive and portable biosensor for detecting Emetic B.cereus is constructed for visual quantitative detection of Emetic B.cereus. The method described in the invention and the matching portable device can also be extended to portable detection of other pathogenic microorganisms.

[0120] Therefore, the present invention adopts the above-mentioned paper-based chip sensor for detecting emetic Bacillus cereus based on aptamer constant temperature amplification, which can complete the detection within 1 hour, has the advantages of visualized quantitative detection, portability, high sensitivity, etc., and provides a research basis for the instant detection of emetic Bacillus cereus. It can solve the problems in the current detection methods that rely on large-scale instruments and professional technicians, the experiments are complicated, the time is long, the efficiency is low, and there is no efficient immunological detection method for Emetic B.cereus, which cannot meet the needs of rapid and portable detection of Emetic B.cereus in food.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A paper-based chip sensor for detecting vomiting-type Bacillus cereus based on aptamer constant temperature amplification, characterized in that: Includes a dark box and a microfluidic paper-based chip; The dark box includes a box body, a dark box top cover, a camera, an LED light group, a sample slot and a small power bank; The microfluidic paper-based chip includes a recognition element, a conversion element and a signal output element; The recognition element refers to a nucleic acid aptamer of Bacillus cereus immobilized on a paper-based chip through the interaction of biotin and streptavidin, wherein the Bacillus cereus aptamer is obtained by screening through the Cell-SELEX technology; The conversion element converts the EmeticB.cereus signal into a pH signal through a constructed super sandwich hybridization probe. The signal catalyzes urea to generate ammonia due to the accumulation of urease and triggers a 1:N colorimetric signal output; The signal output element is to use the camera in the dark box to take pictures of the microfluidic paper-based chip in the sample slot and perform imaging processing, and then connect to the smart phone through a data cable to obtain the picture and perform RGB value analysis, and finally obtain the concentration of the target bacteria in combination with the standard curve; The recognition element is a nucleic acid aptamer fixed on a paper substrate, and the aptamers are DL-1 and DL-2, and their sequences are shown in SEQ ID NO.1-2.

2. The paper-based chip sensor according to claim 1, characterized in that: The transformation element is a super sandwich hybridization probe, the composition sequence is DL-1-YC, UM-DL, DL-4, and the sequence is shown in SEQ ID NO.4-6.

3. Application of the paper-based chip sensor according to any one of claims 1 to 2 in the detection of vomiting-type Bacillus cereus, characterized in that: The application is not intended for disease diagnosis.

4. The detection method of the paper-based chip sensor according to claim 1, characterized in that: The following steps are involved: Step 1: Add the sample solution and N-UM-DL-1 droplets to the circular area on the microfluidic paper chip and wait for the reaction for 25 minutes; The preparation method of N-UM-DL-1 solution is as follows: DL-NH is coupled to UM-DNA with urease through MBS, DL-4 and DL-1-YC are added, and 2+ Under the catalysis of , the nucleic acid aptamer N-UM-DL-1 connected to N ureases was obtained; Step 2: Wash with ddH2O for 5 min; Step 3: Add urea phenol red indicator at pH ~ 5 and react for 30 minutes to observe the color change of the paper-based chip; Step 4: Place the microfluidic paper-based chip into the sample slot of the dark box, connect the smartphone to the data cable of the dark box, obtain the image and RGB value information, and determine the concentration of Emetic B.cereus based on the standard curve of R / G value.

5. Use of the detection method as claimed in claim 4 in the detection of vomitogenic Bacillus cereus in food.

6. The use according to claim 5, characterized in that: The food is a high-starch rice and flour product, including rice and rice noodles.

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