CN115109679A - Bacterial high-throughput quantitative detection system based on deoxyribozyme and application thereof - Google Patents
Bacterial high-throughput quantitative detection system based on deoxyribozyme and application thereof Download PDFInfo
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Abstract
The invention discloses a bacterial high-throughput quantitative detection system based on deoxyribozyme and application thereof, belonging to the technical field of biological detection. The system comprises: the device comprises a power supply module, a pressure supply module, a microfluidic droplet generation module, a fluorescence detection module and a photoelectric conversion module. The micro-fluidic droplet generation module blends the water phase containing bacteria and the oil phase to realize single bacteria wrapping, and after the bacteria wrapping, the micro-fluidic droplet generation module carries out short-time incubation to finish target object identification; the liquid drop with bacteria will generate strong fluorescence signal, which is detected by fluorescence detector and transmitted to computer via photoelectric converter for counting bacteria. The system adopts a micro-droplet reaction mode, highly integrates each step, reduces the errors of manual operation and experiment, more importantly improves the detection speed and flux, and makes up the defects of the traditional culture method; the accuracy of qualitative and quantitative determination is improved by combining a novel molecular biological method; the device has simple structure and is easy to popularize and use in a laboratory.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a bacterial high-throughput quantitative detection system based on deoxyribozyme and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
At present, pathogenic bacteria become a great threat to public health, and high-throughput detection of bacteria is a key to realizing effective control of pathogenic bacteria. Currently, a number of pathogenic bacteria are found clinically, which severely threaten human health and the ecosystem. Therefore, the development of a rapid and high-throughput detection technology for pathogenic bacteria has great significance in the aspects of realizing pollution control, further preventing antibiotic abuse, preventing the formation of multi-drug resistant bacteria, clinical treatment guidance and the like.
The current bacteria detection method mainly comprises a culture method and a gene detection method. The culture method is a gold-labeled method, but is limited by sensitivity, the method needs to culture a collected sample for a long time to obtain a relatively large amount of surviving bacteria to meet the accuracy of qualitative and quantitative analysis, has the inherent defects of long period (1-2 days), complicated sample pretreatment steps and the like, and is difficult to meet the current clinical requirements. In recent years, a variety of emerging molecular biology techniques based on gene detection have been developed, such as quantitative pcr (filmarray bcid), synthetic biology (CRISPR), sequencing (Oxford Nanopore MinION), and some emerging biosensing methods. Many bacteria identification methods can shorten the detection time to several hours and have high detection flux, but for low-concentration bacteria (1-100 CFU/mL), the culture and enrichment are still required for a long time, and the detection flux is limited. In addition, these methods typically require expensive equipment and complex sample pre-treatment (e.g., cell lysis, nucleic acid extraction) to purify and enrich for the target, and purification can result in loss of target, further reducing detection sensitivity. Therefore, establishing a bacterial analysis method with high sensitivity and high flux is a key for solving the problem of rapid bacterial analysis, and establishing a corresponding analysis system is also a basis for realizing detection.
The dnazyme is a nucleic acid molecule fragment with a special structure and function obtained by in vitro screening, i.e., Exponential Enrichment, of ligand phylogenetic Evolution (SELEX), and mainly comprises an aptamer with a specific recognition target substance, a dnazyme with a catalytic ability, an aptamer ribozyme with a recognition and catalytic function of a target substance, and the like. Among them, the biological recognition function of deoxyribozymes is very similar to that of antibodies, and has many advantages of wide target range, high stability, convenience for chemical modification and synthesis, etc. After the deoxyribozyme is bonded with a fluorescent molecule, a molecular beacon can be formed and used for detecting a target object. Therefore, the molecular recognition method is rapidly developed for biological sensing, is widely applied to recognition and detection of nucleic acid, protein, bioactive molecules and disease markers, and shows good application prospect.
At present, most of high-flux and rapid detection methods for pathogenic bacteria are gene analysis methods, genotype analysis can only be completed, phenotype analysis cannot be realized, and development of novel and high-flux pathogenic bacteria phenotype analysis methods and detection systems for obtaining more detailed drug resistance information is of great significance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a bacterial high-throughput quantitative detection system based on deoxyribozyme and application thereof, wherein the system comprises: the device comprises a power supply module, a pressure supply module, a micro-fluidic liquid drop generation module, a fluorescence detection module and a photoelectric conversion module. The micro-fluidic droplet generation module mixes the water phase containing bacteria with the oil phase to realize single-bacteria wrapping, and after the bacteria wrapping, the droplet generation module carries out short-time incubation to finish target object identification; the diameter of a micro channel in the micro-fluidic chip module is equivalent to that of a capillary detection light window, so that only one liquid drop passes through the detection light window every time when sample introduction is carried out; the fluorescence detection module comprises a fluorescence detector; the photoelectric conversion module comprises a data acquisition unit and a data processor, bright fluorescence can be generated when liquid drops with bacteria exist, the reason is that a fluorescent group and a quenching group are simultaneously bonded on deoxyribozyme (RFD-EC), when bacteria do not exist, the RFD-EC is in a fluorescence quenching state, and a lower fluorescence detection background is formed under the irradiation of laser; when the bacteria specific secretory protein is detected, the deoxynucleotide chain of the RFD-EC is cut into a short chain, the distance between a fluorescent group (F) and a quenching group (Q) in a molecule is increased, fluorescence can be recovered under the irradiation of exciting light, a strong fluorescent signal is generated, the fluorescent signal is detected by a fluorescence detector, and the fluorescent signal is transmitted to a computer through a photoelectric conversion device for counting bacteria.
The technical scheme of the invention is as follows:
a bacterial high-flux quantitative detection system based on deoxyribozyme comprises a pressure supply module, a dropping liquid production module, a detection module and a photoelectric conversion module which are connected in sequence; the pressure supply module comprises a gas supply device, a pressure pump and a sample bottle group which are connected in sequence, the sample bottle group is connected with the droplet generation module, and the sample group comprises an oil phase sample bottle and a water phase sample bottle which are connected in parallel between the pressure pump and the droplet generation module; a flow sensor a is arranged between the water phase sample bottle and the dropping liquid production module, and a flow sensor b is arranged in front of the oil sample bottle and the dropping liquid production module; the liquid drop generation module comprises a micro-fluidic chip, the micro-fluidic chip comprises a water sample inlet, an oil sample inlet, an outlet, and a flow channel for connecting the inlet and the outlet, the flow channel for connecting the water sample inlet and a channel for connecting the oil sample inlet are crossed before reaching the outlet, a water phase sample bottle is connected with the water sample inlet, the oil sample bottle is connected with the oil sample inlet, the outlet is connected with one end of a capillary tube, and the other end of the capillary tube is connected with the detection module.
Further, in the above technical solution, the gas supply device includes a nitrogen cylinder or an air compressor; the gas flow rate of the gas supply device is 10-100 nL/min.
Further, in the technical scheme, the inner diameter of the capillary tube is 25-100 μm, and the length of the capillary tube is 10-200 cm; the droplets in the capillary are oil-in-water or water-in-oil, and each droplet contains a bacterium.
Further, in the above technical solution, the microfluidic chip is provided with a water sample inlet, an oil sample inlet, an outlet, and a water sample flow channel connecting the water sample inlet and the outlet, and an oil sample flow channel connecting the oil sample inlet and the outlet, wherein the water sample inlet, the oil sample inlet, and the water sample outlet are on the same straight line; the oil sample flow channel is branched into two parallel flow channels by being close to an oil sample inlet, and the two parallel flow channels are intersected with the water sample flow channel in front of the outlet and are connected to the outlet together; or the water sample flow channel is branched into two parallel flow channels by being close to the water sample inlet, and the two parallel flow channels are intersected with the oil sample flow channel before the outlet and are connected to the outlet together.
Further, in the above technical scheme, the dropping liquid production module includes a temperature control system, and the temperature control system controls the temperature of the microfluidic chip and the capillary.
Further, in the above technical solution, the detection module includes a fluorescence detector, and a light source of the fluorescence detector irradiates onto the transparent light window of the capillary for exciting the fluorescence-labeled target protein.
Further, in the above technical scheme, the photoelectric conversion module includes a signal collector and a data processor, and the fluorescence signal is received by the fluorescence detector and then transmitted to the data processor through the signal collector, so as to quantitatively count the number of bacteria.
The pressure supply module is connected with the power module.
The data processor comprises a computer; the power module is a direct current power supply.
Further, in the above technical solution, the bacteria include klebsiella pneumoniae and escherichia coli.
Further, in the above technical scheme, the aqueous phase sample bottle comprises a bacterial sample, a buffer solution, and a deoxyribozyme solution; the bacterial sample comprises urine, sputum or feces; the oil phase sample bottle comprises 3M TM Novec TM 7500 fluorinated oil.
When the bacteria is klebsiella pneumoniae, the bacterial sample comprises sputum; when the bacteria is escherichia coli, the bacterial sample comprises urine, sputum or feces.
Further, in the above technical solution, the dnazyme solution includes dnazyme chains as shown in SEQ ID No.1 and SEQ ID No. 2.
The invention also provides application of the system in high-throughput quantitative detection of bacteria.
Compared with the prior art, the system has the following advantages:
1. the system has simple structure and low cost, and is easy to use in other laboratories.
2. The system can realize on-line and integrated wrapping of single bacteria, realize rapid reaction in a micro-reaction system, and greatly improve the detection speed and the detection flux.
3. The online and highly integrated system reduces the error caused by manual operation in the whole process, and is favorable for improving the accuracy of quantification
Drawings
FIG. 1 is a schematic structural diagram of a bacterial high-throughput quantitative detection system based on deoxyribozyme.
In the figure, 1, a gas supply device; 2. a pressure pump; 3. an oil phase sample bottle; 4. a water phase sample bottle; 5. a flow sensor b; 6. a flow sensor a; 7. an air inlet pipe b; 8. a liquid outlet pipe b; 9. an air inlet pipe a; 10. a liquid outlet pipe a; 11. a microfluidic chip; 12. a water sample inlet; 13. an oil sample inlet; 14. An outlet; 15. a capillary tube; 16. a temperature control system; 17. a detection module; 18. a photoelectric conversion module; 19. an oil sample flow channel; 20. a water sample flow passage.
FIG. 2 is an image of a monolayer drop in application example 1 of the present invention, and the drop is photographed under a fluorescence microscope (the bright spot in the drop represents Klebsiella pneumoniae).
Detailed Description
For a better understanding of the present invention, the present invention is illustrated by examples and application examples. The present invention is not intended to be limited to the particular embodiments and examples shown.
TABLE 1 nucleic acid sequences for use in the invention
Example 1
The invention provides a system for quantitatively detecting protein in a single cell, which comprises: the power supply module is used for supplying power to the whole system;
the pressure supply module is used for supplying pressure to the system and comprises a pressure supply device and a flow sensor;
the micro-fluidic droplet generation module is used for wrapping single bacteria and controlling the single bacteria, comprises a chip substrate, a chip upper cover, a sample inlet pipe (two phases are respectively an oil phase and a water phase), a sample outlet pipe and a micro-channel, and the chip upper cover is covered on the chip substrate etched with the micro-channel to form the micro-channel; the chip is schematically shown in FIG. 1, wherein the diameter of the microchannel is 50 μm, the material of the chip cover is PDMS, the material of the chip substrate is quartz, and the section of the microchannel is circular; a polytetrafluoroethylene tube is used as a sample inlet tube and an outlet tube, one end of the sample inlet tube is connected with the inlet end of the chip microchannel, the other end of the sample inlet tube is inserted into a test tube (water phase) filled with the bacterial suspension, one end of the other sample inlet tube is connected with the inlet end of the chip microchannel, and the other end of the other sample inlet tube is inserted into a test tube filled with an oil phase and is connected in a sealing way; one end of the sample outlet pipe is connected with the outlet end of the chip micro-channel, the other end of the sample outlet pipe is connected with a reducing two-way, the other end of the reducing two-way is connected with a quartz capillary tube with the outer diameter of 365 mu m, and the inner diameter of the quartz capillary tube is 50 mu m; the liquid drop generation module also comprises a temperature control and resistance wire;
the pressure supply module pumps the oil phase sample and the water phase sample into the oil sample inlet and the water sample inlet respectively, the oil phase sample and the water phase sample are mixed at the intersection of the water sample flow passage and the oil smoke flow passage to form water-in-oil micro-droplets, the micro-droplets in the chip enter the capillary after passing through the two-way variable diameter passage, the rapid reaction and the effective identification are completed in the capillary, and the capture and the marking of target single bacteria are completed; finally, the fluorescence intensity is measured and counted through a detection light window in the detection module;
the fluorescence detection module comprises a fluorescence detector, the wavelength of a light source of the fluorescence detector is 450nm, and the spectral response range of the photoelectric amplifier is 320-1100 nm;
a photoelectric conversion module comprising: the data collector is Sepu3010, the data processor is computer, the light source of the fluorescence detector irradiates on the transparent capillary light window, the diameter of the light window is 50 μm, the fluorescence molecule irradiated by the light source emits fluorescence, the fluorescence is received by the detector, then the fluorescence is transmitted to the computer through Sepu3010, the number of the positive micro-liquid drops is judged according to the fluorescence threshold, and the number of bacteria is calculated through Gaussian distribution.
The working sequence of the modules is as follows: 1. the power supply module is started to supply power to the whole system; 2. the pressure supply module supplies pressure to the system; 3. the micro-fluidic chip liquid drop generating module forms micro-liquid drops; 4. the liquid drops flow through the capillary, and the reaction and the biological recognition are rapidly completed in the capillary; 5. the fluorescence detection module carries out quantitative detection on fluorescence; 6. the photoelectric conversion module counts bacteria.
Application example 1 Rapid determination of Klebsiella pneumoniae Using RFD-EC recognition molecules and bacterial enumeration
RFD-EC recognition molecules are used for rapidly determining kanamycin-resistant Klebsiella pneumoniae, and the specific operation is as follows:
a) culturing engineering bacteria of kanamycin-resistant Klebsiella pneumoniae, adding 10 mu L kanamycin-resistant bacteria into 5mL SOB culture medium, and carrying out overnight culture at the temperature of 37 ℃ on a shaking table and the shaking speed of 150 rpm;
b) after overnight incubation, the concentration of klebsiella pneumoniae was measured using a microplate reader, and when OD600 was 0.5, the bacteria were removed from the shaker at a concentration of about 10 8 CFU/mL；
c) Diluting kanamycin-resistant Klebsiella pneumoniae with sterile water to make the concentration of the Klebsiella pneumoniae in 10 3 ～10 5 CFU/mL, the number of bacteria in 105 μ L bacteria sample is respectively 100, 1000, 10000 bacteria;
d) the bacteria were centrifuged at 8000rpm for 10min (4 ℃), and resuspended in RB solution containing 60mmol/L HEPES, 130mmol/L NaCl, 25mmol/L MgCl 2 And a liquid-transfering gun is adopted to repeatedly suck and beat uniformly;
e) adopting FAM and Dabcyl molecules to carry out fluorescence labeling on the SEQ ID NO.2 molecules to form deoxynucleotides shown in SEQ ID NO. 2;
f) mixing SEQ ID NO.1 and SEQ ID NO.2 to form RFD-EC recognition molecules;
g) adding 105 μ L of antibiotic (with concentration of 32 μ g/mL), 105 μ L of Klebsiella pneumoniae, 30 μ L of RFD-EC recognition molecules (including 25 μ L of 100 μ M SEQ ID NO.1 and 5 μ L of 10 μ M SEQ ID NO.2) into a test tube, operating and measuring fluorescence by using the system according to the steps in example 1, wrapping bacteria into micro-droplets by using a water-in-oil type droplet microfluidic chip, optimizing the concentration of the bacteria so that the number of the bacteria wrapped in each micro-droplet is less than or equal to 1, setting the excitation wavelength to be 450nm, matching the emission wavelength with the excitation and emission wavelengths of the fluorescently-labeled molecules and the reference solution, and measuring the fluorescence intensity.
FIG. 2 is an image of a single layer droplet in this example, which is a photograph taken under a fluorescence microscope (the bright spot in the droplet represents Klebsiella pneumoniae). As can be seen from FIG. 2, under the microscope, the bacteria are clearly observed to be wrapped by the liquid drops, and obvious fluorescent bright spots are shown.
SEQUENCE LISTING
<110> university of Large Community
<120> bacterial high-throughput quantitative detection system based on deoxyribozyme and application thereof
<130> 2022
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<170> PatentIn version 3.5
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ctatgaactg actratgacc tcactaccaa g 31
Claims (10)
1. A bacterial high-throughput quantitative detection system based on deoxyribozyme is characterized in that: the device comprises a pressure supply module, a liquid dropping production module, a detection module and a photoelectric conversion module which are connected in sequence; the pressure supply module comprises a gas supply device (1), a pressure pump (2) and a sample bottle group which are connected in sequence, the sample bottle group is connected with the droplet generation module, and the sample group comprises an oil phase sample bottle (3) and a water phase sample bottle (4) which are connected in parallel between the pressure pump (2) and the droplet generation module; a flow sensor a (6) is arranged between the water phase sample bottle (4) and the dropping liquid production module, and a flow sensor b (5) is arranged in front of the oil sample bottle (3) and the dropping liquid production module; the liquid drop generation module comprises a micro-fluidic chip (11), the micro-fluidic chip (11) comprises a water sample inlet (12), an oil sample inlet (13), an outlet (14) and a flow channel connecting the inlet and the outlet, the flow channel connecting the water sample inlet and a channel connecting the oil sample inlet are intersected before reaching the outlet (14), a water phase sample bottle (4) is connected with the water sample inlet (12), an oil sample bottle (3) is connected with the oil sample inlet (13), the outlet (14) is connected with one end of a capillary tube (15), and the other end of the capillary tube (15) is connected with a detection module (17).
2. The bacterial high-throughput quantitative detection system according to claim 1, characterized in that: the gas supply device (1) comprises a nitrogen cylinder or an air compressor; the gas flow rate of the gas supply device (1) is 10-100 nL/min.
3. The bacterial high-throughput quantitative detection system according to claim 1, characterized in that: the inner diameter of the capillary tube (15) is 25-100 mu m, and the length is 10-200 cm; the droplets in the capillary (15) are oil-in-water or water-in-oil, each droplet containing a bacterium.
4. The bacterial high-throughput quantitative detection system according to claim 1, characterized in that: a water sample inlet (12), an oil sample inlet (13), an outlet (14), a water sample flow channel (20) for connecting the water sample inlet (12) and the outlet (14), and an oil sample flow channel (19) for connecting the oil sample inlet (13) and the outlet (14) are arranged on the micro-fluidic chip (11), wherein the water sample inlet (12), the oil sample inlet (13) and the outlet (14) are on the same straight line; the oil sample flow channel (19) is branched into two parallel flow channels from a position close to the oil sample inlet (13), and the two parallel flow channels are intersected with the water sample flow channel (20) in front of the outlet (14) and are connected to the outlet (14) together; or the water sample flow channel (20) is branched into two parallel flow channels from the position close to the water sample inlet (12), and the two parallel flow channels are intersected with the oil sample flow channel (19) in front of the outlet (14) and are connected to the outlet (14) together.
5. The bacterial high-throughput quantitative detection system according to claim 1, characterized in that: the dropping production module comprises a temperature control system (16), wherein the temperature control system (16) controls the temperature of the microfluidic chip (11) and the capillary (15).
6. The bacterial high-throughput quantitative detection system according to claim 1, characterized in that: the detection module (17) comprises a fluorescence detector, and a light source of the fluorescence detector irradiates on a transparent light window of the capillary tube and is used for exciting the fluorescence-labeled target protein;
the photoelectric conversion module (18) comprises a signal collector and a data processor, and the fluorescence signal is received by the fluorescence detector and then transmitted to the data processor through the signal collector to quantitatively count the number of bacteria.
7. The bacterial high-throughput quantitative detection system according to claim 1, characterized in that: the bacteria include Klebsiella pneumoniae and Escherichia coli.
8. The bacterial high-throughput quantitative detection system according to claim 1, characterized in that: the water phase sample bottle (4) comprises a bacterial sample, a buffer solution and a deoxyribozyme solution; the bacterial sample comprises urine, sputum or feces; the oil phase sample bottle (3) comprises 3M TM Novec TM 7500A fluorinated oil.
9. The bacterial high-throughput quantitative detection system according to claim 1, characterized in that: the deoxyribozyme solution comprises deoxynucleotide chains shown as SEQ ID NO.1 and SEQ ID NO. 2.
10. Use of the system of any one of claims 1-9 for high throughput quantitative detection of bacteria.
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| CN114062679A (en) * | 2021-11-16 | 2022-02-18 | 中国科学院上海微系统与信息技术研究所 | Single-cell secretion high-throughput detection method and system based on droplet microfluidics |
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