Microorganism detection method and device thereof

Abstract

The invention provides a microbiological detection method and a microbiological detection device, and relates to the technical field of biological detection. The microbiological detection method comprises the following steps: modifying a dendritic nucleic acid skeleton to the surface of a magnetic bead through a biotin-streptavidin system to form an aptamer-magnetic bead-peptide nucleic acid skeleton complex; the aptamer-magnetic bead-peptide nucleic acid skeleton complex is applied to microbiological detection based on a micro-fluidic chip. The microbiological detection method disclosed by the invention can achieve the technical effects of rapidness, high sensitivity, high selectivity, low cost and full-automatic microbiological detection, and the corresponding microbiological detection device has the characteristics of portability, wide generalization and low professional requirements on operators.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a microorganism detection method and a device thereof. Background Art

[0002] With the widespread use of traditional Chinese medicine products, ensuring their safety has become an important issue. The 2023 edition of the Chinese Pharmacopoeia stipulates the control of bacteria inspection for non-sterile solid oral preparations containing raw medicinal materials, mainly targeting bacteria such as Escherichia coli, Salmonella, and bile-resistant Gram-negative bacteria. The presence of these bacteria may cause harm to human health, especially living microorganisms can cause large-scale pollution in a short period of time due to their rapid reproduction and environmental resistance. At the same time, endotoxins released after the death or lysis of microorganisms can also cause serious health risks and even threaten public safety.

[0003] In the prior art, the control bacteria detection method specified in the Chinese Pharmacopoeia is the microbial counting method. Although this method has high sensitivity and good accuracy, it takes a long time to detect and has strict environmental requirements, making it unsuitable for rapid on-site detection. In addition, although immunology and nucleic acid amplification-based technologies have high specificity and sensitivity, they cannot distinguish between live and dead bacteria. Although ATP bioluminescence technology can achieve quantitative and immediate detection of live microorganisms, it cannot specifically identify target microorganisms.

[0004] Therefore, the TCM industry is in urgent need of a detection method with fast detection speed, high sensitivity, high specificity, simple operation and low cost to achieve timely diagnosis of a variety of live microorganisms in the complex system of TCM. Summary of the invention

[0005] In view of the above problems, the present invention provides a microorganism detection method to solve at least one problem existing in the prior art.

[0006] To achieve the above-mentioned purpose, in the first aspect, the present invention provides a composition, which is an aptamer-magnetic bead-DNA backbone complex, wherein the aptamer-magnetic bead-DNA backbone complex is an immunomagnetic bead modified with multiple dendritic structures; wherein the carboxyl group of the aptamer sequence is covalently bonded to the amino group of the DNA backbone in the magnetic bead-DNA backbone complex; the biotin that modifies the DNA backbone is combined with the avidin that modifies the magnetic beads; wherein the DNA backbone is a DNA backbone with a U base introduced into the 2' amino group.

[0007] In a second aspect, the present invention provides a kit for detecting microorganisms, the kit comprising the composition as described above.

[0008] In a third aspect, the present invention provides a method for preparing a composition, the method comprising:

[0009] A biotin-modified DNA backbone is introduced on the surface of avidin-modified immunomagnetic beads; wherein the DNA backbone is a DNA backbone with a U base introduced into the 2' amino group; a carboxyl-modified microbial aptamer sequence is introduced by covalently bonding the carboxyl group of the aptamer sequence to the amino group of the DNA backbone; and an aptamer-magnetic bead-DNA backbone complex having a plurality of dendritic backbones centered on the immunomagnetic beads is formed.

[0010] In a fourth aspect, the present invention provides a method for detecting microorganisms, the method comprising:

[0011] Introducing a biotin-modified DNA backbone onto the surface of avidin-modified magnetic beads to form a magnetic bead-DNA backbone complex; wherein the DNA backbone is a DNA backbone with a U base introduced into the 2' amino group;

[0012] The carboxyl group of the aptamer sequence is covalently bonded to the amino group of the DNA backbone, and a carboxyl-modified microbial aptamer sequence is introduced into the magnetic bead-DNA backbone complex to form an aptamer-magnetic bead-DNA backbone complex; wherein the end of the microbial aptamer sequence is modified with a fluorescent group FAM; and the end of the DNA sequence complementary to the microbial aptamer sequence is modified with a quenching group TAMRA;

[0013] Injecting the aptamer-magnetic bead-DNA backbone complex into the channel of the microfluidic chip, the immunomagnetic beads form an immunomagnetic bead chain at the main channel of the microfluidic chip under the action of the magnetic sensing structure;

[0014] The sample to be tested is continuously injected into the channel of the microfluidic chip, and when the target microorganism is not present in the sample to be tested, the fluorescent group FAM and the quenching group TAMRA undergo fluorescence resonance energy transfer effect;

[0015] When the target microorganism exists in the sample to be tested, the target microorganism competes with the complementary DNA sequence, the quenching group TAMRA is released, and the fluorescence of the fluorescent group FAM is restored; the total concentration of the target microorganism in the sample to be tested is determined according to the fluorescence intensity.

[0016] Furthermore, preferably, the method further includes introducing a lysis reagent into the channel of the microfluidic chip to release ATP in the target microorganism, and detecting the content of the released ATP based on the ATP bioluminescence principle to determine the concentration of the live target microorganism in the sample to be tested.

[0017] Further, preferably, the above-mentioned microbial detection method is applied to microbial detection of traditional Chinese medicine, and the microbial aptamer is one of the aptamer of Salmonella typhimurium, the aptamer of Escherichia coli, the aptamer of Staphylococcus aureus, and the aptamer of Pseudomonas aeruginosa.

[0018] Furthermore, preferably, the number of channels of the microfluidic chip is 2-8.

[0019] In a fifth aspect, the present invention provides a microfluidic chip for microbial detection, which is applied to the above-mentioned microbial detection method; it includes more than two microfluidic chip channels for carrying immunomagnetic beads.

[0020] Furthermore, preferably, the channels of the microfluidic chip are arranged in a centrally symmetrical manner.

[0021] In a sixth aspect, the present invention provides a device for detecting microorganisms, comprising the microfluidic chip for detecting microorganisms as described above;

[0022] A magnetic sensing structure corresponding to the immunomagnetic beads is arranged below the microfluidic chip.

[0023] The beneficial effects of the present invention are as follows:

[0024] The present invention combines the carboxyl-modified aptamer fragments on the nucleic acid backbone through the amino groups marked on the nucleic acid backbone, that is, the dendritic backbone is modified to the carboxylated magnetic bead surface through the biotin-streptavidin system (BAS); and an aptamer-magnetic bead-DNA backbone complex is formed. The aptamer-magnetic bead-DNA backbone complex is applied to microbial detection. The DNA backbone not only provides a larger surface area and multiple binding sites, but also optimizes the spatial position of the aptamer, effectively avoiding mutual interference between aptamers, thereby enhancing the capture ability of the target bacteria, and achieving the technical effect of significantly improving the sensitivity and specificity of the detection method. In addition, the chemical stability of the nucleic acid main backbone is better than that of antibodies, and is not easily affected by environmental factors such as temperature, pH value, and solvent, thereby achieving the technical effect of enhancing the durability of the immunomagnetic beads and extending the storage time.

[0025] The microbial detection method of the present invention also utilizes the fluorescence resonance energy transfer effect of the two fluorescent groups FAM and TAMRA to inhibit fluorescence, significantly reducing the background signal and effectively reducing the background noise, thereby achieving the technical effect of greatly improving the specificity and sensitivity of the detection. In addition, the design of this fluorescence detection and ATP bioluminescence detection can achieve the simultaneous detection of live target bacteria and total target bacteria, and can also achieve quantitative detection of dead bacteria through calculation.

[0026] The microfluidic chip and microorganism detection device provided by the present invention have the advantages of small size, low cost, easy batch production and carrying. The detection method based on the combination of microfluidic chip technology, magnetic separation technology and ATP bioluminescence technology is simple to operate, and the detection sensitivity and specificity are greatly improved. It can realize the limit inspection of a variety of live microorganisms and is suitable for commercial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 FIG. 1 is a schematic diagram of the structure of a dendritic skeleton of a composition according to an embodiment of the present invention.

[0028] Figure 2 The figure is a schematic diagram of a formation process of a composition according to an embodiment of the present invention.

[0029] Figure 3 The figure is a schematic diagram of the principle of a microorganism detection method according to an embodiment of the present invention.

[0030] Figure 4 The figure is a schematic structural diagram of a microfluidic chip according to an embodiment of the present invention.

[0031] Figure 5 The figure is a schematic diagram of the overall structure of a microorganism detection device according to an embodiment of the present invention.

[0032] Figure 6 The figure is a schematic diagram of the magnetic sensing structure of a microorganism detection device according to an embodiment of the present invention.

[0033] Figure 7 The linear relationship between the detection signal and the concentration of different types of live bacteria according to an embodiment of the present invention.

[0034] in,

[0035] 1. Chip body; 2. Channel entrance; 3. Inlet channel; 4. Main channel; 5. Detection cavity; 6. Exit channel; 7. Channel exit; 8. Serrated iron sheet; 9. NdFeB magnet; 11. Device housing; 12. First to fourth sampling holes; 13. Sample injection tube; 14. Peristaltic pump; 15. Waste liquid tube; 16. Waste liquid barrel. DETAILED DESCRIPTION

[0036] Peptide nucleic acid is a molecule that mimics DNA. The introduction of a U base with an amino group at the 2' position into the DNA backbone can form a peptide nucleic acid (PNA), which is characterized by replacing the sugar-phosphate backbone in DNA with a peptide-like backbone composed of N-(2-aminoethyl)glycine units. This structure gives PNA some unique properties, such as higher affinity and specificity to bind to target DNA or RNA sequences, as well as better stability and resistance to nucleases; PNA with a U base with an amino group at the 2' position can be synthesized, and this modification may enhance the binding ability of PNA to nucleic acids or change its biochemical properties.

[0037] Biotin-Streptavidin System (BAS) is a technology widely used in biomedical research. Its core lies in the extremely high affinity and specific binding between biotin and streptavidin. The affinity of this interaction is very high, and the affinity constant (K) can reach 10^15^mol / L, which is much higher than the affinity between antigen and antibody, making BAS extremely sensitive and specific in detection and quantitative analysis.

[0038] Magnetic nanoparticles (MNPs), or magnetic beads, are a class of nanoscale materials with magnetic properties, usually composed of iron, cobalt, nickel or their oxides. Due to their unique physicochemical properties, such as superparamagnetism, high specific surface area and easy functionalization, MNPs have a wide range of applications in the biomedical field, including drug delivery, magnetic resonance imaging, magnetic hyperthermia, biological separation and detection, etc.

[0039] Microfluidic chip is a scientific technology that manipulates fluids in micro-nanoscale space, also known as Lab-on-a-Chip. It uses micromachining technology to process various microstructure units such as micron to sub-millimeter fluid channels, reaction or detection chambers, filters or sensors on substrates made of silicon, metal, polymer, glass, quartz and other materials.

[0040] The present invention will be described in more detail with reference to the following examples. However, the protection scope of the present invention is not limited to the following examples.

[0041] Example 1

[0042] Figure 1 and Figure 2 The overall structure of the composition is described. Figure 1 Schematic diagram of the structure of a dendritic skeleton of a composition according to an embodiment of the present invention. Figure 2 The present invention is a schematic diagram of a process for forming a composition according to an embodiment of the present invention.

[0043] The present invention provides a composition, which is an aptamer-magnetic bead-DNA skeleton complex; specifically, the DNA skeleton is a DNA skeleton with a U base introduced into the 2' amino group; in a specific implementation process, it can also be a peptide nucleic acid skeleton.

[0044] Specifically, the aptamer-magnetic bead-DNA backbone complex is an immunomagnetic bead modified with multiple dendritic structures; wherein the carboxyl group of the aptamer sequence is covalently bonded to the amino group of the DNA backbone in the magnetic bead-DNA backbone complex; and the biotin that modifies the DNA backbone is bonded to the avidin that modifies the magnetic beads.

[0045] like Figure 1 As shown, the dendritic skeleton includes a peptide nucleic acid skeleton (or a DNA skeleton with a U base introduced into the 2' amino group), biotin modified at the front end of the peptide nucleic acid skeleton, and an aptamer sequence bound to the peptide nucleic acid skeleton. In a specific implementation process, the avidin is streptavidin. The dendritic skeleton is modified onto the surface of the magnetic beads by a biotin-streptavidin system (BAS).

[0046] It should be noted that PNA can be combined with other molecules through chemical modification and coupling technology to form a complex structure. In this embodiment, the carboxyl-modified aptamer fragment is combined with the nucleic acid backbone through the amino group labeled on the nucleic acid backbone, that is, the dendritic backbone is modified to the carboxyl-modified magnetic bead surface through the biotin-streptavidin system (BAS).

[0047] The invention provides a method for preparing a composition, which comprises the following steps.

[0048] S1. Introduce a biotin-modified peptide nucleic acid backbone onto the surface of avidin-modified immunomagnetic beads.

[0049] like Figure 2 As shown, the specific implementation process is to wash the magnetic beads with MEST buffer (10mM MES, containing 0.05% Tween 20, pH6.0) and 2-(N-morpholino)ethanesulfonic acid (MES) respectively; then activate the magnetic beads with 1-(3-dimethylaminopropyl)-3-ethylcarbopolamide (EDC) and N-hydroxysuccinimide (NHS); streptavidin binds to the carboxyl groups on the magnetic beads, and 1.5% bovine serum albumin (BSA) is used to fill the carboxyl vacancies on the magnetic beads that are not bound to streptavidin to obtain MNP-SA. The biotin-modified U base fragment is modified by the amino-labeled nucleic acid fragment to form a complex on MNP-SA.

[0050] That is to say, Figure 2 As shown in the upper part of the picture, the avidin-modified magnetic beads and the biotin-modified DNA backbone are combined through the biotin-streptavidin system, that is, through the binding of biotin and avidin, to obtain a magnetic bead-peptide nucleic acid backbone complex.

[0051] S2. The carboxyl group of the aptamer sequence is covalently bonded to the amino group of the peptide nucleic acid backbone to introduce the carboxyl-modified microbial aptamer sequence, thereby forming an aptamer-magnetic bead-peptide nucleic acid backbone complex with multiple dendritic backbones centered on the immunomagnetic beads.

[0052] like Figure 2As shown in the lower part of the picture, the aptamer sequence is combined with the magnetic bead-peptide nucleic acid backbone complex by covalently bonding the carboxyl group of the aptamer sequence to the amino group of the peptide nucleic acid backbone in the magnetic bead-peptide nucleic acid backbone complex to obtain the aptamer-magnetic bead-peptide nucleic acid backbone complex.

[0053] That is to say, the aptamer of the target microorganism is hybridized with the complex, and the hybridization product is hybridized with the base fragment to obtain the desired magnetic beads. The above composition can be detected by fluorescence, electrochemistry or other detection methods. The magnetic beads themselves can also be used as signal output, for example, by fluorescent labels or enzyme labels on the magnetic beads to achieve signal amplification and detection. Then, by measuring the fluorescence intensity, recording the electrochemical signal or reading the signal of other types of biosensors, quantitative or qualitative analysis of the target molecule can be achieved.

[0054] As an improvement of this embodiment, in order to apply the above-mentioned composition to the scene of microbial detection of microfluidic chips, the microfluidic chip technology is combined with the fluorescence detection technology. The specific implementation process is to hybridize the aptamer of the target microorganism with the FAM group with the complex, and the hybridization product is hybridized with the base fragment with the TAMRA group to obtain the required magnetic beads. That is to say, by utilizing the fluorescence resonance energy transfer effect of the two fluorescent groups of FAM and TAMRA to suppress fluorescence, the background signal is significantly reduced, and the background noise is effectively reduced, thereby achieving a technical effect of greatly improving the specificity and sensitivity of the detection.

[0055] As an improvement of this embodiment, the present invention provides a kit for detecting microorganisms, the kit comprising the above-mentioned composition and a microbial cell viability detection reagent. The magnetic beads obtained by the above-mentioned composition preparation method can be stored in a 4°C refrigerator for 21 days; that is, the present invention only needs to change the corresponding aptamer to prepare the dendritic skeleton by the above-mentioned method, and a new probe can be prepared according to the above-mentioned method. The above-mentioned composition preparation method has the characteristics of simple operation and strong versatility.

[0056] The microbial cell viability detection reagent includes an ATP extraction reagent and a luciferin-luciferase system. The ATP extraction reagent is a buffer solution containing 1% benzalkonium chloride (BAC); the buffer is 25mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes), 2.0mM ethylenediaminetetraacetic acid (EDTA), and the pH is 7.6; the luciferin-luciferase system includes: 1mg / mL luciferin buffer solution; the buffer is 25mM Hepes, 2.0mM EDTA, 20mM magnesium chloride, 1.2mg / mL BSA, 2.0mM DTT, and the pH is 7.6; 5mg / mL luciferase buffer solution; the buffer is 25mM Hepes, 2.0mM EDTA, 20mM magnesium chloride, 1.2mg / mL BSA, 2.0mM DTT, and the pH is 7.6.

[0057] 1% benzalkonium chloride (BAC) is used as a cationic surfactant in the microbial cell viability detection reagent to ensure the efficient extraction of ATP from trace microorganisms. Combined with the optimized buffer and DTT components, the stability of the reagent is significantly improved. At the same time, the optimized luciferin-luciferase system enhances the intensity of the light signal, thereby improving the sensitivity and specificity of the detection. The kit not only improves the accuracy of the detection, but also shortens the reaction time and reduces operational errors. Due to its low dependence on the operating environment, this detection method can meet the clinical needs of instant and rapid detection, and is suitable for microbial detection in a variety of traditional Chinese medicine products, and has broad application prospects.

[0058] In general, the carboxyl-modified aptamer fragments are bound to the nucleic acid backbone through the amino groups labeled on the nucleic acid backbone, that is, the dendritic backbone is modified to the carboxylated magnetic bead surface through the biotin-streptavidin system (BAS); an aptamer-magnetic bead-peptide nucleic acid backbone complex is formed. The aptamer-magnetic bead-peptide nucleic acid backbone complex is applied to microbial detection. The peptide nucleic acid backbone not only provides a larger surface area and multiple binding sites, but also optimizes the spatial position of the aptamer, effectively avoiding mutual interference between aptamers, thereby enhancing the capture ability of the target bacteria, and achieving the technical effect of significantly improving the sensitivity and specificity of the detection method. In addition, the chemical stability of the nucleic acid main backbone is better than that of antibodies, and is not easily affected by environmental factors such as temperature, pH value, and solvents, thereby achieving the technical effect of enhancing the durability of the immunomagnetic beads and extending the storage time.

[0059] Example 2

[0060] Figure 3 FIG. 2 is a schematic diagram showing the principle of a microorganism detection method according to an embodiment of the present invention. Figure 3 As shown, the present invention provides a method for detecting microorganisms, the method comprising the following steps:

[0061] S110, introducing a biotin-modified peptide nucleic acid backbone onto the surface of avidin-modified magnetic beads to form a magnetic bead-peptide nucleic acid backbone complex.

[0062] The peptide nucleic acid backbone may be a DNA backbone having a U base introduced into the 2' amino group.

[0063] The specific implementation process is to wash the magnetic beads with MEST buffer (10mM MES, containing 0.05% Tween 20, pH 6.0) and 2-(N-morpholino)ethanesulfonic acid (MES) respectively; then activate the magnetic beads with 1-(3-dimethylaminopropyl)-3-ethylcarbamide (EDC) and N-hydroxysuccinimide (NHS); streptavidin binds to the carboxyl groups on the magnetic beads, and 1.5% bovine serum albumin (BSA) is used to fill the carboxyl vacancies on the magnetic beads that are not bound to streptavidin to obtain MNP-SA. The biotin-modified U base fragment is modified by the amino-labeled nucleic acid fragment to form a complex on MNP-SA.

[0064] S120, introducing a carboxyl-modified microbial aptamer sequence into the magnetic bead-peptide nucleic acid backbone complex by covalently bonding the carboxyl group of the aptamer sequence to the amino group of the peptide nucleic acid backbone, thereby forming an aptamer-magnetic bead-peptide nucleic acid backbone complex; wherein the end of the microbial aptamer sequence is modified with a fluorescent group FAM; and the end of a DNA sequence complementary to the microbial aptamer sequence is modified with a quenching group TAMRA.

[0065] The biotin-modified U base fragment is modified with an amino-labeled nucleic acid fragment onto MNP-SA to form a complex. In other words, the aptamer of the target microorganism is hybridized with the complex, and the hybridization product is hybridized with the base fragment to obtain the desired magnetic beads.

[0066] S130, injecting the aptamer-magnetic bead-peptide nucleic acid backbone complex into the channel of the microfluidic chip, and the immunomagnetic beads form an immunomagnetic bead chain at the main channel of the microfluidic chip under the action of the magnetic sensing structure.

[0067] Specifically, in a microfluidic chip, the magnetic beads can be aggregated into a chain structure through the action of an external magnetic field. This structure can be used to fix or manipulate the magnetic beads to facilitate subsequent immune responses. The magnetic beads can form a chain structure under the action of a magnetic field. This structure can increase the contact area between the magnetic beads, thereby improving the efficiency of capturing target molecules. This magnetic bead chain can be used in subsequent detection processes.

[0068] S140, continuously injecting the sample to be tested into the channel of the microfluidic chip, when the target microorganism is not present in the sample to be tested, the fluorescent group FAM and the quenching group TAMRA undergo fluorescence resonance energy transfer effect.

[0069] Specifically, the sample to be tested is injected into the microfluidic chip, and the sample contains target molecules that may bind to the aptamers on the magnetic beads. In this embodiment, the target molecules are target microorganisms. The aptamers on the magnetic beads specifically bind to the target molecules in the sample to form immune complexes. These complexes can be manipulated by the magnetic field to achieve separation from unbound target molecules.

[0070] S150. When the target microorganism exists in the sample to be tested, the target microorganism competes with the complementary DNA sequence, the quenching group TAMRA is released, and the fluorescence of the fluorescent group FAM is restored; the total concentration of the target microorganism in the sample to be tested is determined according to the fluorescence intensity.

[0071] The fluorescence resonance energy transfer effect of the two fluorescent groups FAM and TAMRA is used to inhibit fluorescence, significantly reducing the background signal and effectively reducing the background noise, thereby achieving the technical effect of greatly improving the specificity and sensitivity of detection.

[0072] Specifically, the base fragment with the TAMRA group is a base fragment paired with the end of the target microbial aptamer. When the TAMRA group and the FAM group are gathered between 1-10nm, fluorescence energy resonance transfer will occur and fluorescence quenching will occur. When the distance between the two is greater than 10nm, the fluorescent group can emit fluorescence when irradiated with excitation light.

[0073] As an improvement of this embodiment, in order to achieve simultaneous detection of live target bacteria and total target bacteria, quantitative detection of dead bacteria can also be achieved through calculation. The above fluorescence detection is combined with the design of ATP bioluminescence detection to obtain the following scheme.

[0074] S160, introducing a lysis reagent into the channel of the microfluidic chip to release ATP in the target microorganism, and detecting the content of the released ATP based on the ATP bioluminescence principle to determine the concentration of the live target microorganism in the sample to be tested.

[0075] The binding force between the target microorganism aptamer and the target microorganism is greater than the binding force between the base fragment paired with the end thereof.

[0076] As an improvement of this embodiment, the above-mentioned microbial detection method is applied to microbial detection of traditional Chinese medicine. The microbial aptamer is one of the aptamer of Salmonella typhimurium, the aptamer of Escherichia coli, the aptamer of Staphylococcus aureus, and the aptamer of Pseudomonas aeruginosa. The microbial detection method is applied to microbial detection of traditional Chinese medicine, and has the technical effects of fast detection speed, high sensitivity, high specificity, simple operation, and low cost.

[0077] In order to improve the detection efficiency of the above-mentioned microbial detection method in the microbial detection scenario of traditional Chinese medicine, the number of channels of the microfluidic chip is set to 2-8. In other words, multiple channels are set in the microfluidic chip to achieve simultaneous detection of multiple microorganisms. Taking the setting of 4 channels as an example, based on the above-mentioned microbial detection method, the detection efficiency is further improved by 4 times.

[0078] The present invention can quickly detect the microbial content in a sample within 2 hours by using advanced technologies such as microfluidic magnetic separation, nucleic acid detection and ATP bioluminescence reaction. Compared with traditional culture methods, this detection method based on microfluidic chips not only has a faster detection speed, but also has lower technical requirements for operators and is easier to popularize and apply. Compared with immunological and molecular biological detection methods, the present invention achieves specific detection of live microorganisms by integrating ATP bioluminescence technology.

[0079] Example 3

[0080] The present invention provides a microfluidic chip for microbial detection, which is applied to the above-mentioned microbial detection method; it includes more than two microfluidic chip channels for carrying immunomagnetic beads. The microfluidic chip channels are centrally symmetrically arranged. Specifically, a plurality of lofting holes are arranged at the center position of the chip body 1, and each lofting hole corresponds to a microfluidic chip channel. In other words, a plurality of microfluidic chip channels are radiated outward from a central point, that is, they are in a centrally symmetrical divergent structure.

[0081] An exemplary description is given below with the number of microfluidic chip channels being 4. Figure 4 FIG. 1 is a schematic diagram of the structure of a microfluidic chip of this embodiment. Figure 4 As shown, the microfluidic chip includes a chip channel for carrying immunomagnetic beads and a chip body 1; 4 microfluidic chip channels are arranged on the chip body 1, and 4 lofting holes are arranged at the center of the chip body 1. The lofting holes are cylindrical, and each lofting hole corresponds to a microfluidic chip channel, and the 4 microfluidic chip channels are arranged in a cross. In the specific implementation process, the entrances of the four channels are the same; as an improvement of this embodiment, the chip material on at least one side of the main channel is a visible material. The microfluidic chip provided by the present invention is provided with four detection chambers for different types of target microorganisms, which can detect different live microorganisms at the same time, and has a small size, low cost, and is conducive to mass production. The device containing the microfluidic chip can realize rapid detection of multiple live target microorganisms in the complex system of traditional Chinese medicine, which can greatly simplify the operation steps, and the device is portable. It can realize limit inspection of multiple live microorganisms and is suitable for commercial promotion and application.

[0082] The chip channel includes a channel inlet 2, an inlet channel 3, a main channel 4, a detection cavity 5, an outlet channel 6 and a channel outlet 7 which are arranged in sequence; the channel inlet 2 and the channel outlet 7 are respectively arranged at the two ends of the chip channel; the channel inlet 2 is connected with the inlet channel 3, and the channel outlet 7 is connected with the channel 6; the detection cavity 5 and the main channel 4 are arranged between the inlet channel 3 and the channel 6.

[0083] Wherein, the material of the microfluidic chip is one of PDMS, polymethyl methacrylate PMMA, polypropylene PP, cycloolefin copolymer COC, cycloolefin polymer COP and polycarbonate PC. That is to say, polydimethylsiloxane (PDMS) is preferably selected, and PDMS has the characteristics of excellent biocompatibility, transparency, flexibility, gas permeability, chemical stability and cost-effectiveness. In the specific implementation process, materials such as polypropylene (PP), cycloolefin copolymer (COC), cycloolefin polymer (COP), polymethyl methacrylate (PMMA) and polycarbonate (PC) can be used as alternative materials.

[0084] By setting up a four-channel microfluidic chip, multiple experimental steps are integrated on one chip, reducing contact with the external environment and effectively reducing the risk of contamination. The four-channel design makes it possible to detect four different types of bacteria at the same time, expanding the detection range. When detecting the same bacteria, the four-channel design helps to improve the repeatability and data consistency of experimental results. In addition, the diagonal layout reduces interference between magnetic fields and reduces the mutual influence of the luminescence intensity of different bacteria. The shape of the microfluidic chip is designed to resemble a four-leaf clover, which not only has functional advantages, but also enhances the product's visual effects and brand recognition, and improves market competitiveness.

[0085] A magnetic sensing structure corresponding to the immunomagnetic beads is arranged below the microfluidic chip. The microfluidic chip of the present invention applies microfluidic immunomagnetic separation technology. The immunomagnetic separation technology is a process in which the immunomagnetic beads located in the chip channel and the magnetic sensing structure located below the microfluidic chip are used to quickly separate and enrich the target.

[0086] Figure 5 and Figure 6 The overall structure of the microbial detection device is described. Figure 5 The figure is a schematic diagram of the overall structure of a microorganism detection device according to an embodiment of the present invention. Figure 6 It is a schematic diagram of the structure of the magnetic sensing structure of a microorganism detection device according to an embodiment of the present invention.

[0087] A microorganism detection device is used for automatic detection of multiple microorganisms in a complex Chinese medicine system, comprising the microfluidic chip as described above; the microfluidic chip comprises a chip channel for carrying immunomagnetic beads and a chip body 1; 4 microfluidic chip channels are arranged on the chip body 1, 4 lofting holes are arranged at the center of the chip body 1, the lofting holes are arranged in a cylindrical shape, each lofting hole corresponds to a microfluidic chip channel, and the 4 microfluidic chip channels are arranged in a cross. The chip channel comprises a channel inlet 2, an inlet channel 3, a main channel 4, a detection cavity 5, an outlet channel 6 and a channel outlet 7 arranged in sequence; the channel inlet 2 and the channel outlet 7 are respectively arranged at the two ends of the chip channel; the channel inlet 2 is connected to the inlet channel 3, and the channel outlet 7 is connected to the outlet channel 6; the detection cavity 5 and the main channel 4 are arranged between the inlet channel 3 and the outlet channel 6. A magnetic sensing structure corresponding to the immunomagnetic beads is arranged below the microfluidic chip.

[0088] In order to realize the automated detection of small molecule compound detection devices based on microfluidic chips, such as Figure 5 As shown, the specific implementation may include but is not limited to the following: the small molecule compound detection device based on the microfluidic chip includes a device housing 11, and a detection component disposed in the device housing 11; the detection component includes but is not limited to the first to fourth sample holes 12 for adding reagents, a sample injection tube 13 for connecting the sample holes and the microfluidic chip, and a peristaltic pump 14 is disposed on the sample injection tube 13; it also includes a waste liquid bucket 16 connected to the channel outlet 7 of the microfluidic chip, and a waste liquid bucket 16 and a waste liquid tube 15 for connecting the channel outlet 7 and the waste liquid bucket 16. A power switch is also disposed on the device housing 11.

[0089] The microbial detection device of the present invention makes the access of samples and the discharge of waste liquid more efficient, shortens the operation time, reduces the reagents and sample processing steps, and thus reduces the experimental cost. Since the required sample volume is small, this method improves the efficiency of sample use and is particularly suitable for the detection of rare or difficult-to-obtain samples.

[0090] The device is divided into three modules, namely, a power module, a control module, and a detection module. The power module includes a power supply, a power switch, and a voltage-stabilized power supply. The power supply is used to supply power to the device, and the voltage-stabilized power supply is used to stabilize the voltage of the device. The control module includes a switch, a radiator, a TTL-485, a peristaltic pump, a buzzer, a stepper motor linear guide, a driver, a relay, a single motor, a solenoid valve, a control switch, and a photodiode. The switch is used to control the start-up state of the control module. The peristaltic pump is used to push the liquid through the microfluidic channel. The stepper motor linear guide is used to carry the magnetic grid to move under the chip to control the position of the immune magnetic beads in the chip. The detection module includes a CCD, a converter, and a computer. The CCD is used to convert the color in the detection hole into an optical signal. The converter is used to convert the optical signal in the CCD into an electrical signal. The computer end is used to read the signal.

[0091] To achieve automation, the present apparatus further requires that the local and remote data processing modules may include energy-efficient processors or controllers, and digital memory, such as flash memory, both of which may be used to assist in processing, caching and storing data captured from sensors operably coupled to the frame, such as CCD capture devices (e.g., cameras); and / or acquired and / or processed using remote processing modules and / or remote data repositories, possibly for transmission to a display after such processing or retrieval. The local and remote data processing modules are operably coupled, such as by wired or wireless communication links, to the remote processing modules and remote data repositories, such that these remote modules are operably coupled to each other and can be used as resources for the local and remote data processing modules. In one embodiment, the remote processing modules may include one or more relatively powerful processors or controllers configured to analyze and process data and / or image information. In one embodiment, the remote data repositories may include relatively large-scale digital data storage facilities that may be obtained via the Internet or other network configurations in a "cloud" resource configuration. In one embodiment, all data is stored and all calculations are performed in the local and remote data processing modules, thereby allowing full autonomous use from any remote module.

[0092] like Figure 6As shown, in the specific implementation process, the magnetic induction structure is a magnetic grid separator; the magnetic grid separator includes a magnetic grid shell, a NdFeB magnet 8 and a serrated iron sheet 9; a serrated iron sheet 9 and at least two NdFeB magnets 8 are arranged in the chamber formed by the magnetic grid shell; adjacent NdFeB magnets 8 are mutually repelling and separated by the serrated iron sheet 9. Two adjacent magnets repel each other and are separated by a serrated iron sheet. This structure can ensure that the magnetic field intensity at the tip of the serrated iron sheet is the strongest, and the immunomagnetic beads gather at the tip of the serrated iron sheet and form a vertical chain, increasing the contact area between the immunomagnetic beads and the sample, and improving the capture and enrichment efficiency. Further, the grade of the ultra-thin square NdFeB magnet is N52, and the rubidium boron magnet is a square sheet with a side length of 8-12 mm and a thickness of 0.5-1.5 mm; the serrated iron sheet is a square sheet with the same side length as the rubidium boron magnet and a thickness of 0.4-0.6 mm. Preferably, the side length of the neodymium boron magnet is 10 mm, and the thickness is 1 mm; the side length of the serrated iron sheet is 10 mm, and the thickness is 0.5 mm, and the serration interval is 1 mm. By limiting the shape of the magnet and the iron sheet according to the size of the specific magnetic grid separator, a more accurate magnetic field can be provided for the immunomagnetic beads.

[0093] In the specific implementation process, the main channel is designed as a hexagonal structure; the inlet channel and the outlet channel form an acute angle with the main channel respectively; wherein the inlet channel and the outlet channel form an angle of 20°-40° with the main channel respectively; in order to further reduce the sample residue in the channel; preferably, the angle is 30°. The width of the main channel is 0.8-1.2㎝, and the depth is 150-250μm; the width of the inlet channel and the outlet channel is 0.5-1㎝, and the depth is 150-250μm. Preferably, the main channel is 2cm long, 1cm wide, and 200μm high; the width of the inlet channel and the outlet channel is 0.5-1mm, and the depth is 200μm.

[0094] Example 4

[0095] Taking the detection of Salmonella typhimurium, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in a complex traditional Chinese medicine system as an example, the above-mentioned microbial detection device was used for detection.

[0096] Among them, the microfluidic chip consists of a 40mm×40mm glass base plate and a PDMS cover plate, and the glass base plate and the PDMS cover plate are bonded together by plasma.

[0097] A microorganism detection method of the present invention comprises steps S11-S17.

[0098] S11. Introduce a biotin-modified peptide nucleic acid backbone onto the surface of avidin-modified magnetic beads to form a magnetic bead-peptide nucleic acid backbone complex.

[0099] The peptide nucleic acid backbone may be a DNA backbone having a U base introduced into the 2' amino group.

[0100] The specific implementation process is to wash the magnetic beads with MEST buffer (10mM MES, containing 0.05% Tween 20, pH 6.0) and 2-(N-morpholino)ethanesulfonic acid (MES) respectively; then activate the magnetic beads with 1-(3-dimethylaminopropyl)-3-ethylcarbamide (EDC) and N-hydroxysuccinimide (NHS); streptavidin binds to the carboxyl groups on the magnetic beads, and 1.5% bovine serum albumin (BSA) is used to fill the carboxyl vacancies on the magnetic beads that are not bound to streptavidin to obtain MNP-SA. The biotin-modified U base fragment is modified by the amino-labeled nucleic acid fragment to form a complex on MNP-SA.

[0101] S12. The carboxyl group of the aptamer sequence is covalently bonded to the amino group of the peptide nucleic acid backbone, thereby introducing a carboxyl-modified microbial aptamer sequence into the magnetic bead-peptide nucleic acid backbone complex to form an aptamer-magnetic bead-peptide nucleic acid backbone complex; wherein the end of the microbial aptamer sequence is modified with a fluorescent group FAM; and the end of a DNA sequence complementary to the microbial aptamer sequence is modified with a quenching group TAMRA.

[0102] The biotin-modified U base fragment is modified with an amino-labeled nucleic acid fragment onto MNP-SA to form a complex. In other words, the aptamer of the target microorganism is hybridized with the complex, and the hybridization product is hybridized with the base fragment to obtain the desired magnetic beads.

[0103] The specific implementation process is as follows: the magnetic beads are washed with MEST buffer (10 mM MES, containing 0.05% Tween 20, pH 6.0) and 2-(N-morpholino)ethanesulfonic acid (MES), respectively; the magnetic beads are then activated with 1-(3-dimethylaminopropyl)-3-ethylcarbopolamide (EDC) and N-hydroxysuccinimide (NHS); streptavidin binds to the carboxyl groups on the magnetic beads, and 1.5% bovine serum albumin (BSA) is used to fill the carboxyl vacancies on the magnetic beads that are not bound to streptavidin. Finally, the supernatant is removed by magnetic recovery, and washed three times with PBS buffer, and a nucleic acid backbone with a repeating sequence modified with biotin and amino-modified U bases is added, the reaction is carried out for 1 hour, magnetic recovery is carried out for 3 minutes, the supernatant is removed, and the carboxyl-functionalized aptamer is added to react in 0.1 M MES buffer (pH 6.0) for 2 hours. The carboxyl functionalized aptamer solution was pre-activated with ethyl-3-(3-(dimethylamino)propyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). After that, the mixture was centrifuged for 10 minutes, washed with PBS, resuspended with 1 mL of PBS solution, and a DNA (1uM) sequence with a quenching group TAMRA at the end was added, and PBS was added to 1 mL, reacted at 37°C for 35 minutes, and the supernatant was removed by magnetic recovery, and washed three times with PBS solution to obtain the desired magnetic beads.

[0104] Among them, the target microorganism aptamers are aptamers of Salmonella typhimurium, aptamers of Escherichia coli, aptamers of Staphylococcus aureus, and aptamers of Pseudomonas aeruginosa.

[0105] S13, injecting the aptamer-magnetic bead-peptide nucleic acid backbone complex into the channel of the microfluidic chip, and the immunomagnetic beads form an immunomagnetic bead chain at the main channel of the microfluidic chip under the action of the magnetic sensing structure.

[0106] Specifically, the aptamer-magnetic bead-peptide nucleic acid backbone complex can be stored in a refrigerator at 4°C for 21 days. During the test, the aptamer-magnetic bead-peptide nucleic acid backbone complex is first injected into the chip channel from the inlet hole, and the immunomagnetic beads in the main channel can form an immunomagnetic bead chain under the high gradient magnetic field formed by the magnetic grid below.

[0107] S14, continuously injecting the sample to be detected into the channel of the microfluidic chip, so that the target microorganism in the sample binds to the target microorganism aptamer on the magnetic bead chain, irradiating the detection cavity with a 494nm laser, irradiating the detection cavity with a fluorimeter, and converting the optical signal into a corresponding value.

[0108] Specifically, the first sample hole is pre-placed with diluted target microorganism standard solutions of different concentrations, which flow through the magnetic bead chain under the action of the peristaltic pump, and the target microorganisms in the solution are captured and enriched, and other interfering substances flow into the waste liquid bucket with the solution. Then, the cleaning solution pre-placed in the third sample hole flows through the microfluidic channel under the action of the peristaltic pump to flush the residual sample matrix to reduce interference.

[0109] S15. Mix 500 μL of 1% benzalkonium chloride (BAC) buffer solution, 100 μL of 25 mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) with a pH of 7.6, and 2.0 mM ethylenediaminetetraacetic acid (EDTA) to prepare an ATP extraction reagent; mix 50 μL of 1 mg / mL luciferin buffer solution, 25 mM Hepes buffer with a pH of 7.6, 2.0 mM EDTA buffer, 20 mM magnesium chloride, 1.2 mg / mL BSA, 2.0 mM DTT, and 100 μL of 5 mg / mL luciferase buffer solution to prepare a luciferin-luciferase solution system; mix the ATP extraction reagent and the luciferin-luciferase solution system to prepare a microbial cell viability detection reagent, and immediately pass it into the channel of the microfluidic chip to generate a bioluminescent reaction.

[0110] Among them, 1% benzalkonium chloride (BAC) is used as a cationic surfactant in the microbial cell viability detection reagent to ensure the efficient extraction of ATP from trace microorganisms. Combined with the optimized buffer and DTT components, the stability of the reagent is significantly improved. At the same time, the optimized luciferin-luciferase system enhances the intensity of the light signal, thereby improving the sensitivity and specificity of the detection. The kit not only improves the accuracy of the detection, but also shortens the reaction time and reduces operational errors.

[0111] S16. Start the stepper motor to drive the magnetic grid to move to the bottom of the chip detection cavity. Under the action of the magnetic field, the magnetic bead-target microorganism complex is concentrated in the detection cavity.

[0112] S17. The microbial cell viability detection reagent is introduced into the channel of the microfluidic chip to generate a bioluminescent reaction. Finally, the relative luminescent intensity is detected by a bioluminescent detector. The converter converts the light signal into a corresponding numerical value. At this time, the instrument buzzes and alarms, indicating that the detection is over. Then the computer software is connected for reading. The buzzer sounds again when the reading is over. Then the instrument starts to automatically clean the microfluidic chip channel. After the cleaning is completed, the instrument automatically stops. At this time, the magnetic grid stays on the right side of the slide rail, that is, below the detection cavity of the chip. When the next detection starts, the magnetic grid is reset and automatically moves to the bottom of the chip channel.

[0113] An example of repeated steps is as follows: the magnetic beads modified with the target microorganism antibody are injected into the chip channel from the inlet again, and the magnetic beads in the main channel can form a magnetic bead chain under the high gradient magnetic field formed by the magnetic grid below; then the honey pill sample liquid pre-placed in the second sampling hole flows through the magnetic bead chain under the action of the peristaltic pump, the target microorganism in the sample is captured and enriched, and other interfering substances enter the waste liquid barrel with the liquid flow; then the cleaning solution pre-placed in the third sampling hole flows through the microfluidic channel under the action of the peristaltic pump to flush the residual sample matrix to reduce interference. Then start the stepper motor to drive the magnetic grid to move to the bottom of the chip detection cavity. Under the action of the magnetic field, the magnetic bead-target microorganism complex is concentrated in the detection cavity, and the detection cavity is irradiated with a 494nm laser. The detection cavity is irradiated with a fluorimeter, and the converter converts the light signal into a corresponding value. Then, the microbial cell viability detection reagent in the fourth sampling hole is introduced into the chip channel to cause a bioluminescent reaction. Finally, the relative luminescence intensity is detected using a bioluminescence detector. The converter converts the light signal into a corresponding value. At this time, the instrument buzzes and an alarm sounds, indicating that the detection is over. Then connect to the computer software for reading. The buzzer sounds again when the reading is over, and then the instrument starts to automatically clean the microfluidic chip channel. After the cleaning is completed, the instrument automatically stops. At this time, the magnetic grid stays on the right side of the slide rail, that is, below the detection cavity of the chip. When the next detection starts, the magnetic grid is reset and automatically moves to the bottom of the chip channel.

[0114] The above steps are repeated to detect the Chinese medicine samples to obtain the corresponding values ​​and draw a standard curve. Figure 7 The linear relationship between the detection signal and concentration of different types of live bacteria according to an embodiment of the present invention is shown. Figure 7As shown, the regression equation for live Salmonella detection is y=0.6324X-0.2773, R2 is 0.9739; the regression equation for live Escherichia coli detection is y=0.5952X-0.08928, R2 is 0.9673; the regression equation for live Staphylococcus aureus is y=0.6248X-0.4265, R2 is 0.9698; the regression equation for live Pseudomonas aeruginosa is y=0.6619X-0.4252, R2 is 0.9590. Moreover, the time to complete the detection of the target microbial amount in the honey pill is less than 2h. The device using the microfluidic chip can realize rapid, highly sensitive, highly selective, low-cost, and fully automatic detection of live Salmonella, live Escherichia coli, live Staphylococcus aureus, and live Pseudomonas aeruginosa, and the device is portable and widely popularizable, and has low professional requirements for operators, which provides a more portable and rapid detection method for detecting live microorganisms in honey pills. The present invention provides an application of the above-mentioned method for detecting multiple live microorganisms based on a microfluidic chip in the process of detecting the amount of multiple live microorganisms in honey pills. The above-mentioned method for detecting multiple live microorganisms based on a microfluidic chip is suitable for the detection of multiple live microorganisms in various scenarios, and is particularly suitable for on-site rapid detection with high requirements for detection sensitivity and high timeliness.

[0115] The device using the microfluidic chip can realize rapid, high-sensitivity, high-selectivity, low-cost, and fully automatic detection of multiple microorganisms in complex traditional Chinese medicine systems. The device is portable and widely applicable, and has low professional requirements for operators, which provides a more portable and rapid detection method for detecting multiple microorganisms in complex traditional Chinese medicine systems.

[0116] The sources of the reagents used are as follows: 2-(N-morpholino)ethanesulfonic acid (MES) buffer was provided by Beijing Bairuiji Biotechnology Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbopolamide (EDC) and N-hydroxysuccinimide (NHS) were provided by Maclean; MEST was provided by Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; 1% benzalkonium chloride (BAC) solution was provided by Sigma-Aldrich, USA; 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) was provided by Genview, USA; ethylenediaminetetraacetic acid (EDTA) was provided by Shandong Yinuo Chelate Chemical Co., Ltd.; luciferin and luciferase were from Promega, USA; magnesium chloride was provided by Beijing Beihua Chemical Pharmaceutical Co., Ltd.; dithiothreitol (DTT) and bovine serum albumin (BSA) were provided by Beijing Bailingwei Technology Co., Ltd. FAM and TAMRA were provided by Meige Biotechnology Co., Ltd. Other reagents were imported and domestic analytical reagents.

[0117] Among the various experimental supplies mentioned in this article (including but not limited to: chemical reagents, biological products, cells, organisms, instruments, etc.), for those that are special or difficult to obtain, the manufacturers, references or detailed preparation methods have been indicated in the article; those not otherwise specified are conventional experimental supplies and can be easily obtained by various means (such as purchase, self-preparation, etc.) before the date of this application.

[0118] Although the present invention has been described in detail above by general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications and improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A composition, characterized in that The composition is an aptamer-magnetic bead-DNA backbone complex, and the aptamer-magnetic bead-DNA backbone complex is an immunomagnetic bead modified with multiple dendritic structures; The carboxyl group of the aptamer sequence is covalently bonded to the amino group of the DNA skeleton in the magnetic bead-DNA skeleton complex; the biotin modified DNA skeleton is bonded to the avidin modified magnetic bead; and the DNA skeleton is a DNA skeleton with a U base introduced into the 2' amino group.

2. A kit for detecting microorganisms, characterized in that: The kit comprises the composition of claim 1.

3. A method for preparing a composition, characterized in that: Methods include, Introducing a biotin-modified DNA backbone onto the surface of avidin-modified immunomagnetic beads; wherein the DNA backbone is a DNA backbone with a U base introduced into the 2' amino group; The carboxyl group of the aptamer sequence is covalently bonded to the amino group of the DNA backbone to introduce a carboxyl-modified microbial aptamer sequence; an aptamer-magnetic bead-DNA backbone complex with multiple dendritic backbones centered on the immunomagnetic beads is formed.

4. A method for detecting microorganisms, characterized in that: Methods include, Introducing a biotin-modified DNA backbone onto the surface of avidin-modified magnetic beads to form a magnetic bead-DNA backbone complex; wherein the DNA backbone is a DNA backbone with a U base introduced into the 2' amino group; The carboxyl group of the aptamer sequence is covalently bonded to the amino group of the DNA backbone, and a carboxyl-modified microbial aptamer sequence is introduced into the magnetic bead-DNA backbone complex to form an aptamer-magnetic bead-DNA backbone complex; wherein the end of the microbial aptamer sequence is modified with a fluorescent group FAM; and the end of the DNA sequence complementary to the microbial aptamer sequence is modified with a quenching group TAMRA; Injecting the aptamer-magnetic bead-DNA backbone complex into the channel of the microfluidic chip, the immunomagnetic beads form an immunomagnetic bead chain at the main channel of the microfluidic chip under the action of the magnetic sensing structure; The sample to be tested is continuously injected into the channel of the microfluidic chip, and when the target microorganism is not present in the sample to be tested, the fluorescent group FAM and the quenching group TAMRA undergo fluorescence resonance energy transfer effect; When the target microorganism exists in the sample to be tested, the target microorganism competes with the complementary DNA sequence, the quenching group TAMRA is released, and the fluorescence of the fluorescent group FAM is restored; the total concentration of the target microorganism in the sample to be tested is determined according to the fluorescence intensity.

5. The microorganism detection method according to claim 4, characterized in that: The method also includes introducing a lysis reagent into the channel of the microfluidic chip to release ATP in the target microorganism, and detecting the content of the released ATP based on the ATP bioluminescence principle to determine the concentration of the live target microorganism in the sample to be tested.

6. The microorganism detection method according to claim 4, characterized in that: Applicable to microbial detection of traditional Chinese medicine; the microbial aptamer is one or more of the aptamer of Salmonella typhimurium, the aptamer of Escherichia coli, the aptamer of Staphylococcus aureus, and the aptamer of Pseudomonas aeruginosa.

7. The microorganism detection method according to claim 4, characterized in that: The number of channels of the microfluidic chip is 2-8.

8. A microfluidic chip for microbial detection, characterized in that: The microbial detection method as claimed in claim 4; It includes more than two microfluidic chip channels for carrying immunomagnetic beads.

9. The microfluidic chip for microorganism detection according to claim 8, characterized in that: The channels of the microfluidic chip are arranged in a centrally symmetrical manner.

10. A device for detecting microorganisms, characterized in that: A microfluidic chip for microbial detection comprising the microfluidic chip of claim 8; A magnetic sensing structure corresponding to the immunomagnetic beads is arranged below the microfluidic chip.

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