Micro-fluidic chip and method for detecting food-borne pathogenic bacteria

Abstract

The invention provides a micro-fluidic chip and a method for detecting food-borne pathogenic bacteria. The micro-fluidic chip comprises at least one detection area, and the detection area comprises a treatment tank used for collecting and cracking bacterial thalli in a liquid sample to be detected; a component for splitting bacteria thalli is pre-buried in the first pre-buried area; a component for detecting bacterial nucleic acid through loop-mediated isothermal amplification is pre-buried in a reaction tank in the second pre-buried area; wherein the treatment tank is respectively communicated with the reaction tanks in the first pre-buried area and the second pre-buried area through fluid channels. The micro-fluidic chip is based on an LAMP means and a micro-fluidic technology, through a special structural design, only a sample to be detected needs to be homogenized, subsequent nucleic acid component collection, splitting decomposition and detection can be completed in the micro-fluidic chip, manual operation is greatly reduced, and aerosol pollution is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a microfluidic chip and method for detecting bacteria, especially foodborne pathogenic bacteria. Background Art

[0002] Foodborne diseases caused by foodborne pathogenic bacteria are the main problems faced by food safety. They are often accompanied by symptoms such as fever, nausea, vomiting, and diarrhea, and can even lead to death in severe cases. According to the investigation of the WTO, there are approximately 600 million cases of foodborne diseases occurring globally every year, and 420,000 people die as a result.

[0003] With the acceleration of the pace of life and consumers' emphasis on nutrition and health, the pre-cut fruit and vegetable market has become a new trend and new track in the convenient food industry, with a market scale of nearly 3 trillion yuan. Pre-cut fruits and vegetables have promoted the rapid growth of their consumer market due to characteristics such as less nutrient loss and convenient instant consumption. However, at the same time, due to their rich nutrition and tissue damage, they are more vulnerable to microbial contamination. Macroscopically, it is manifested as browning of fruits and vegetables and abnormal odors, and microscopically, it is manifested as a large number of foodborne pathogenic bacteria breeding, etc., resulting in many quality and safety hazards in pre-cut fruits and vegetables.

[0004] Currently, there are various methods applied to the detection of foodborne pathogenic bacteria, such as bacterial culture methods, immunological methods, and molecular biology methods, etc. Among them, the molecular biology method is increasingly widely used in the rapid diagnosis of foodborne pathogenic bacteria due to its advantages such as strong specificity and high sensitivity. However, the nucleic acid extraction from plant tissues is more difficult than that from other samples. The currently common extraction method is manual extraction using a centrifugal column type kit. The quality of the obtained nucleic acid is better than that of other methods, but there are still problems such as low yield and possible inhibition of downstream reactions. In addition, due to the rich content of a large number of secondary metabolites such as polysaccharides and polyphenols in pre-cut fruits and vegetables, it greatly affects the quality and purity of DNA, and in severe cases, it cannot even be used as a template for PCR amplification. The existing conventional detection methods have many adverse factors such as cumbersome steps, long manual operation time, increased possibility of aerosol contamination, and inability to meet the requirements of rapid on-site real-time detection of a large number of samples.

[0005] Based on the current status of foodborne pathogenic bacteria detection, there is an urgent need to develop a functionally integrated microfluidic chip with a simple structure, strong anti-interference ability, and capable of realizing simple operation and accurate detection for application in POCT molecular detection. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies existing in the prior art and provide a microfluidic chip and method for detecting foodborne pathogenic bacteria. Based on the LAMP method and microfluidic technology, the present invention designs a microfluidic chip for detecting foodborne pathogenic bacteria, which only needs to homogenize the sample to be tested, and subsequent nucleic acid collection, lysis and detection can be completed within the microfluidic chip, greatly reducing manual operations and aerosol contamination, and can be applied to the rapid, efficient and simple direct amplification detection of foodborne pathogenic bacteria, especially foodborne pathogenic bacteria in pre-cut fruits and vegetables (such as fresh-cut fruits).

[0007] After research, the inventors of the present invention found that the loop-mediated isothermal amplification (LAMP) technology has the advantages of simple operation, strong specificity, low requirements for instrument equipment and strong anti-interference ability compared with the PCR technology. Combining it with the microfluidic chip technology with the advantages of integration, miniaturization, low cost and high efficiency, and selecting reagents with anti-interference ability can effectively detect the presence of foodborne pathogenic bacteria in pre-cut fruits and vegetables. Therefore, the inventors of the present invention further verified through a large number of experiments that the pre-cut fruit and vegetable liquid sample can be centrifugally enriched first, while collecting as many bacterial cells as possible and reducing the contamination and interference of other components in the sample, that is, while ensuring the collection of the bacteria to be detected, leaving less secondary metabolites such as polysaccharides and polyphenols; the sample is then rapidly lysed by the bacterial lysate to complete the release of bacterial nucleic acids; loop-mediated isothermal nucleic acid amplification is carried out in a microfluidic chip pre-embedded with freeze-dried powder of reagents for detecting target bacteria, thereby completing the detection of bacteria.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] In a first aspect, the present invention provides a microfluidic chip for detecting foodborne pathogenic bacteria, which includes at least one detection area, and the detection area includes:

[0010] A treatment pool for collecting and lysing bacterial cells in the liquid sample to be tested;

[0011] A first pre-embedded area in which components for lysing bacterial cells are pre-embedded;

[0012] A second pre-embedded area in which components for detecting bacterial nucleic acids by loop-mediated isothermal amplification are pre-embedded in the reaction pool;

[0013] Wherein, the treatment pool is communicated with the reaction pools in the first pre-embedded area and the second pre-embedded area via fluid channels.

[0014] According to some embodiments of the present invention, wherein the detection area further includes:

[0015] The first waste liquid pool, which is communicated with the treatment pool via a fluid channel. The first waste liquid pool is used for storing the waste liquid generated after the bacterial collection treatment of the liquid sample to be tested, such as the sterile polysaccharide- and polyphenol-containing waste liquid of the homogenized liquid sample of pre-cut fruits and vegetables to be tested.

[0016] According to some embodiments of the present invention, wherein the detection area further includes:

[0017] The third embedded area, in which a medium for sealing the first waste liquid pool is embedded. The medium can isolate the waste liquid generated after the bacterial collection treatment of the liquid sample to be tested in the first waste liquid pool. In the present invention, the medium is preferably (low melting point) solid paraffin, and the solid paraffin can block the first waste liquid pool after melting.

[0018] According to some embodiments of the present invention, wherein the treatment pool is fluidly communicated with the reaction pool in the second embedded area via a hydrophobic barrier channel. When using a microfluidic chip for detection, when the centrifugal speed is less than the critical speed, the sample liquid containing bacterial nucleic acid cannot enter the second embedded area through the hydrophobic barrier channel; when the centrifugal speed is greater than the critical speed, the sample liquid containing bacterial nucleic acid can enter the second embedded area through the hydrophobic barrier channel.

[0019] According to some embodiments of the present invention, wherein the width of the hydrophobic barrier channel is less than 100 microns; the critical speed is 3000 revolutions per minute.

[0020] According to some specific embodiments of the present invention, wherein the treatment pool is fluidly communicated with the reaction pool in the second embedded area via a hydrophobic barrier channel, and the width of the hydrophobic barrier channel is less than 100 microns, so that when the centrifugal speed is less than 3000 revolutions per minute, the liquid sample (the liquid containing bacterial nucleic acid) cannot enter the second embedded area; when the centrifugal speed is greater than 3000 revolutions per minute, the liquid sample can enter the second embedded area.

[0021] According to some embodiments of the present invention, wherein the second embedded area includes at least two reaction pools, and components for detecting different types of bacteria by loop-mediated isothermal amplification are embedded in different reaction pools. Setting multiple reaction pools in the second embedded area of the microfluidic chip can achieve the simultaneous detection of multiple target bacteria (such as foodborne pathogenic bacteria) with a single sample addition, which is fast, simple, synchronous and efficient. In addition, the second embedded area may further include a reaction pool embedded with a negative control.

[0022] According to some embodiments of the present invention, by weight parts, the components pre-embedded in the first pre-embedded area for lysing bacterial cells include 0.5 - 3 parts of Polyvidone-40 (PVP-40), 0.01 - 0.1 part of Sodium Dodecyl Sulfate (SDS), and 1 - 5 parts of Triton-100. Preferably, by weight parts, the components pre-embedded in the first pre-embedded area for lysing bacterial cells include 1.5 parts of Polyvidone-40, 0.03 part of Sodium Dodecyl Sulfate, and 2 parts of Triton-100.

[0023] According to some embodiments of the present invention, the component for lysing bacterial cells is a bacterial lysate, which contains 0.5 - 3% by weight of PVP-40, 0.01 - 0.1% by weight of SDS, 1 - 5% by weight of Triton-100, and the balance is sterile water. Preferably, the component for lysing bacterial cells is a bacterial lysate, which contains 1.5% by weight of PVP-40, 0.03% by weight of SDS, 2% by weight of Triton-100, and the balance is sterile water. Further preferably, the bacterial lysate may further contain 50 - 100 mM, such as 150 mM of NaCl and 0.5 - 1.5 mM, such as 1 mM of EDTA.

[0024] According to some embodiments of the present invention, a capsule filled with the component for lysing bacterial cells (bacterial lysate) is pre-embedded into the first pre-embedded area. Preferably, the material of the capsule can be aluminum foil. The breaking pressure can be adjusted by regulating the hot-pressing sealing time of the aluminum foil by an aluminum foil sealer. For example, it can be adjusted so that it will not break when the centrifugal speed is less than 2000 revolutions per minute, while when the centrifugal speed is greater than 2000 revolutions per minute, the capsule is subjected to a greater compressive force and ruptures, releasing the bacterial lysate therein.

[0025] In the present invention, when the centrifugal speed is relatively low (such as 1500 revolutions per minute), the bacterial cells in the homogenized liquid sample to be tested (pre-cut fruit and vegetable sample) are collected in the processing pool; when the centrifugal speed is relatively high (2500 revolutions per minute), the processing pool provides a place for the component for lysing bacterial cells (bacterial lysate) released from the first pre-embedded area to lyse the bacterial cells in the liquid sample to be tested.

[0026] According to some embodiments of the present invention, wherein, the detection area further includes:

[0027] A sample adding hole, which is in fluid communication with the treatment pool via a fluid channel; the sample adding hole is used for adding a liquid sample, and the added liquid can be a homogenized liquid sample to be tested (such as a fresh-cut fruit liquid sample). When the microfluidic chip is placed in a chip adapter instrument for low-speed centrifugation (for example, the centrifugation speed is 1500 - 2000 revolutions per minute), the liquid sample to be tested is made to enter the treatment pool;

[0028] A quantitative pool, which is arranged between the treatment pool and the reaction pool in the second pre-embedded area, and is in fluid communication with the treatment pool via a first hydrophobic barrier channel and in fluid communication with the reaction pool in the second pre-embedded area via a second hydrophobic barrier channel; the quantitative pool is used for storing and quantifying the liquid sample to be tested, and can ensure a fixed reaction volume;

[0029] A second waste liquid pool, which is in fluid communication with the reaction pool in the second pre-embedded area via a fluid channel; the second waste liquid pool is used for storing the excess amplification reaction solution; and / or

[0030] A vent hole, which is used to ensure smooth sample addition.

[0031] In the present invention, the hydrophobic barrier channel includes a first hydrophobic barrier channel and a second hydrophobic barrier channel. The quantitative pool is in fluid communication with the first hydrophobic barrier channel and the second hydrophobic barrier channel respectively.

[0032] In the present invention, homogenizing the sample to be tested means cutting up and mixing evenly all of the sample to be tested (such as fresh-cut fruit), putting it into a sterile homogenization bag and performing sufficient homogenization to obtain the liquid sample to be tested.

[0033] Based on the current situation of foodborne pathogenic bacteria in the field of food safety, the inventors of the present invention, through a large number of investigations, selected five foodborne pathogenic bacteria, namely Salmonella (Sm), Staphylococcus aureus (Sa), Listeria monocytogenes (Lm), Escherichia coli O157: H7 (O157), which are the four pathogenic bacteria indicators that must be detected for ready-to-eat pre-cut fruit and vegetable products, and Cronobacter (Cro), which has a relatively high lethality rate for special populations, as the detection targets. Efficient and accurate detection of these foodborne pathogenic bacteria is of extremely important significance for improving the rapid detection ability of foodborne pathogenic bacteria in the food-related industries, especially the pre-cut fruit and vegetable industry.

[0034] Thus, according to some embodiments of the present invention, the components for detecting bacterial nucleic acids by loop-mediated isothermal amplification include a primer set for detecting foodborne pathogenic bacteria, a freeze-dried mixture of a DNA loop-mediated isothermal amplification reaction solution and a fluorescent dye, wherein the DNA loop-mediated isothermal amplification reaction solution contains a DNA polymerase, deoxynucleoside triphosphates, a magnesium ion solution, and a reaction buffer.

[0035] According to some embodiments of the present invention, the primer set for detecting foodborne pathogenic bacteria includes one or more primer sets selected from the following: a primer set for detecting Salmonella, a primer set for detecting Staphylococcus aureus, a primer set for detecting Listeria monocytogenes, a primer set for detecting diarrheagenic Escherichia coli, a primer set for detecting Cronobacter, and a primer set for detecting the bacterial 16S rRNA gene.

[0036] According to some preferred embodiments of the present invention, the primer set for detecting Salmonella includes:

[0037] Outer primer pair Sm-F3 and Sm-B3 (Sm-F3 / B3), whose nucleotide sequences are shown in SEQ ID NO. 1 and 2 respectively; and

[0038] Inner primer pair Sm-FIP and Sm-BIP (Sm-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NO. 3 and 4 respectively;

[0039] The primer set for detecting Staphylococcus aureus includes:

[0040] Outer primer pair Sa-F3 and Sa-B3 (Sa-F3 / B3), whose nucleotide sequences are shown in SEQ ID NO. 5 and 6 respectively; and

[0041] Inner primer pair Sa-FIP and Sa-BIP (Sa-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NO. 7 and 8 respectively;

[0042] The primer set for detecting Listeria monocytogenes includes:

[0043] Outer primer pair Lm-F3 and Lm-B3 (Lm-F3 / B3), whose nucleotide sequences are shown in SEQ ID NO. 9 and 10 respectively; and

[0044] Inner primer pair Lm-FIP and Lm-BIP (Lm-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NO. 11 and 12 respectively;

[0045] The primer set for detecting diarrheagenic Escherichia coli includes:

[0046] Outer primer pair O157-F3 and O157-B3 (O157-F3 / B3), the nucleotide sequences of which are shown in SEQ ID NO. 13 and 14 respectively; and

[0047] Inner primer pair O157-FIP and O157-BIP (O157-FIP / BIP), the nucleotide sequences of which are shown in SEQ ID NO. 15 and 16 respectively;

[0048] The primer set for detecting Cronobacter includes:

[0049] Outer primer pair Cro-F3 and Cro-B3 (Cro-F3 / B3), the nucleotide sequences of which are shown in SEQ ID NO. 17 and 18 respectively; and

[0050] Inner primer pair Cro-FIP and Cro-BIP (Cro-FIP / BIP), the nucleotide sequences of which are shown in SEQ ID NO. 19 and 20 respectively;

[0051] The primer set for detecting bacterial 16S rRNA includes:

[0052] Outer primer pair 16S rRNA-F3 and 16S rRNA-B3 (16S rRNA-F3 / B3), the nucleotide sequences of which are shown in SEQ ID NO. 21 and 22 respectively; and

[0053] Inner primer pair 16S rRNA-FIP and 16S rRNA-BIP (16S rRNA-FIP / BIP), the nucleotide sequences of which are shown in SEQ ID NO. 23 and 24 respectively.

[0054] According to some embodiments of the present invention, the molar ratio of the outer primer pair Sm-F3 / B3 to the inner primer pair Sm-FIP / BIP is 1:3 to 9, preferably 1:8; the molar ratio of the outer primer pair Sa-F3 / B3 to the inner primer pair Sa-FIP / BIP is 1:3 to 9, preferably 1:8; the molar ratio of the outer primer pair Lm-F3 / B3 to the inner primer pair Lm-FIP / BIP is 1:3 to 9, preferably 1:8; the molar ratio of the outer primer pair O157-F3 / B3 to the inner primer pair O157-FIP / BIP is 1:3 to 9, preferably 1:8; the molar ratio of the outer primer pair Cro-F3 / B3 to the inner primer pair Cro-FIP / BIP is 1:3 to 9, preferably 1:8; the molar ratio of the outer primer pair 16S rRNA-F3 / B3 to the inner primer pair 16S rRNA-FIP / BIP is 1:3 to 9, preferably 1:8.

[0055] In the present invention, each set of primers for target bacteria respectively includes a pair of outer primers and a pair of inner primers, and the molar ratio between the primers in each pair of primers is 1:1. For example, in the outer primer pair O157-F3 / B3 for the target of Escherichia coli O157 causing diarrhea, the molar ratio of O157-F3 to O157-B3 is 1:1, and in the inner primer pair O157-FIP / BIP, the molar ratio of O157-FIP to O157-BIP is 1:1.

[0056] In the present invention, the sets of primers are respectively designed and screened according to the invA gene sequence of Salmonella (MK017932), the spa gene sequence of Staphylococcus aureus (X61307), the hylA gene sequence of Listeria monocytogenes (KJ504151), the rfbE gene sequence of Escherichia coli O157: H7 (LC743564) causing diarrhea, the ompA gene of Cronobacter (DQ000206), and the sequence of bacterial 16S ribosomal RNA (947777) in the NCBI database.

[0057] In a second aspect, the present invention provides a method for preparing a microfluidic chip according to the first aspect of the present invention, which includes:

[0058] (1) Dissolving the components for detecting bacterial nucleic acid by loop-mediated isothermal amplification into sterile water to obtain a solution;

[0059] (2) Spotting the solution into the reaction pools in the second pre-embedded area of the microfluidic chip and freeze-drying;

[0060] (3) After freeze-drying, pre-embedding the components for lysing bacterial cells in the first pre-embedded area.

[0061] According to some embodiments of the present invention, wherein the preparation method further includes:

[0062] Pre-embedding the medium for sealing the first waste liquid pool in the third pre-embedded area of the microfluidic chip.

[0063] According to some embodiments of the present invention, wherein the preparation method further includes:

[0064] Covering the top film on the top of the microfluidic chip and performing thermal compression bonding.

[0065] According to some embodiments of the present invention, the preparation method includes:

[0066] (1) Respectively dissolving the sets of primers for detecting bacterial nucleic acid, the DNA loop-mediated isothermal amplification reaction solution, and the fluorescent dye into sterile water to obtain a solution;

[0067] (2) Spotting the solution into the reaction pools in the second pre-embedded area of the microfluidic chip and freeze-drying;

[0068] After freeze-drying, capsules containing components for lysing bacterial cells (bacterial lysate) are pre-embedded in the first pre-embedding area, and low-melting-point solid paraffin is pre-embedded in the third pre-embedding area;

[0069] (4) Cover the top of the microfluidic chip with a plastic top film and perform thermocompression bonding.

[0070] In a third aspect, the present invention provides a method for detecting foodborne pathogenic bacteria, the method comprising performing nucleic acid detection of foodborne pathogenic bacteria using the microfluidic chip according to the first aspect of the present invention.

[0071] According to some preferred embodiments of the present invention, wherein the foodborne pathogenic bacteria are selected from one or more of Salmonella (Sm), Staphylococcus aureus (Sa), Listeria monocytogenes (Lm), Escherichia coli O157: H7 (O157), and Cronobacter (Cro).

[0072] According to some embodiments of the present invention, wherein the method comprises sequentially performing bacterial cell collection and lysis and loop-mediated isothermal amplification reaction on a homogenized liquid sample to be tested using the microfluidic chip according to the first aspect of the present invention, and determining the presence of bacteria in the sample to be tested based on the generated fluorescence curve.

[0073] According to some embodiments of the present invention, the temperature of the loop-mediated isothermal amplification reaction is 60°C to 65°C, preferably 62°C.

[0074] According to some embodiments of the present invention, the time of the loop-mediated isothermal amplification reaction is 30 to 60 minutes, preferably 40 minutes.

[0075] According to some embodiments of the present invention, wherein the method comprises the following steps:

[0076] (1) Add the homogenized liquid sample to be tested to the sample loading hole of the microfluidic chip and centrifuge at 1000 to 1800 revolutions per minute, for example 1500 revolutions per minute, so that the bacterial cells in the liquid sample to be tested are collected in the processing pool;

[0077] (2) Centrifuge and heat at 2000 - 2800 revolutions per minute, for example, 2500 revolutions per minute, to release the components pre-embedded in the first pre-embedded area for lysing bacterial cells, lyse the bacterial cells in the liquid sample to be tested in the treatment pool. Meanwhile, the low-melting-point paraffin pre-embedded in the third pre-embedded area seals the waste liquid generated after the liquid sample to be tested is processed by bacterial collection into the first waste liquid pool;

[0078] (3) Centrifuge at 3000 - 4000 revolutions per minute, for example, 3500 revolutions per minute, so that the bacterial lysate of the liquid sample to be tested enters the reaction pool in the second pre-embedded area, and detect the bacterial nucleic acid by loop-mediated isothermal amplification.

[0079] According to some embodiments of the present invention, wherein the centrifugation in steps (2) and (3) is carried out at 60°C - 65°C, preferably at 62°C.

[0080] According to some embodiments of the present invention, wherein the centrifugation time in step (1) is 1 - 5 minutes, preferably 3 minutes.

[0081] According to some embodiments of the present invention, wherein the centrifugation time in step (2) is 3 - 7 minutes, preferably 5 minutes.

[0082] According to some embodiments of the present invention, wherein the centrifugation time in step (3) is 0.5 - 4 minutes, preferably 2 minutes.

[0083] According to some preferred embodiments of the present invention, wherein the method comprises the following steps:

[0084] Take 200 μL of the homogenized liquid sample of the fruit to be tested and add it to the sample loading hole of the microfluidic chip. Tear off the sealing film, fit it tightly with the sealing film surface facing up. Steadily fix the positioning hole of the microfluidic chip card slot on the tray of the microfluidic nucleic acid analyzer. First, centrifuge at 1500 revolutions per minute for 3 minutes at room temperature, so that all the possible bacterial cells in the liquid sample to be tested are collected into the treatment pool; Secondly, centrifuge at 2500 revolutions per minute for 5 minutes at 62°C, so that the liquid capsule pre-embedded in the first pre-embedded area ruptures, releasing the bacterial lysate, which flows into the treatment pool, where bacterial lysis occurs. Meanwhile, the low-melting-point paraffin pre-embedded in the third pre-embedded area melts during the heating process to seal the waste liquid containing polysaccharides and polyphenols in the liquid sample to be tested into the first waste liquid pool; Thirdly, centrifuge at 3500 revolutions per minute for 2 minutes at 62°C, so that the liquid possibly containing bacterial nucleic acid components flows into the second pre-embedded area; Finally, perform amplification detection at 62°C for 40 minutes, and determine the presence of the bacterial sample according to the change of the fluorescence curve.

[0085] In the present invention, the presence of bacteria is determined based on the amplification results of bacterial internal reference (16S rRNA). When the internal reference (16S rRNA) in the detected fluorescence curve shows an S-shaped curve and any one or more of the target bacteria show an S-shaped curve, it indicates that one or more corresponding bacteria are present in the sample to be tested; when no S-shaped curve appears, it indicates that one or more corresponding bacteria are not present in the sample to be tested.

[0086] When detecting foodborne pathogenic bacteria in pre-cut fruits and vegetables using traditional methods, first, the pre-cut fruits and vegetables (such as fresh-cut fruits) need to be homogenized. Secondly, the liquid after homogenization is taken for high-speed centrifugation for at least 5 minutes, and then the supernatant is discarded. The lower precipitate is taken to extract and purify nucleic acid using a column-type nucleic acid extraction kit. The whole process involves heating, lysis, adsorption, washing, and elution, etc. The steps are relatively cumbersome, and it also involves the introduction of additional chemical reagents (such as binding solution, washing solution, elution solution). There are operations of introducing reagents (such as reagent introduction) and removing waste liquid tubes multiple times, inevitably accompanied by the possibility of aerosol contamination. At the same time, the manual workload is large, and it generally takes a long time (about 1 hour) to obtain purified nucleic acid. After a large number of studies, the inventors of the present invention designed a microfluidic chip for detecting bacteria, especially foodborne pathogenic bacteria, based on the LAMP method and microfluidic technology. This microfluidic chip can greatly reduce the detection steps (such as the steps of adding and removing reagents), and only needs to homogenize the sample of pre-cut fruits and vegetables to be tested and perform fractional centrifugation in the microfluidic chip to complete the whole sample detection process. The manual operation time is less, the probability of aerosol contamination is smaller, and the operation is simple and the detection is accurate.

[0087] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0088] 1. The microfluidic chip provided by the present invention only needs to add and fractionally centrifuge the pre-cut fruits and vegetables sample that has been homogenized to complete the whole sample detection process, realizing rapid, efficient, and simple detection of foodborne pathogenic bacteria in pre-cut fruits and vegetables samples through direct amplification; moreover, the whole detection process is an automated instrument program operation, without the need to take out the microfluidic chip in the middle, without adding samples again and introducing additional reagents, removing multiple manual operations, and reducing the occurrence of aerosol contamination;

[0089] 2. The microfluidic chip and its detection kit provided by the present invention can realize the synchronous detection of the presence of multiple foodborne pathogenic bacteria with a single sample addition, and have the advantages of simple operation, uniform liquid separation, good biocompatibility, and environmental friendliness; in addition, after the microfluidic chip is further designed to have multiple identical detection areas, multiple samples to be tested can be detected simultaneously;

[0090] 3. The microfluidic chip, detection kit, and detection method provided by the present invention can quickly and efficiently achieve the extraction and amplification detection of bacterial nucleic acids, and can directly judge the detection results by detecting the fluorescence signal and the peak time of the fluorescence curve, with simple result determination; in addition, the recognition of 2 pairs of inner primers and outer primers corresponding to each detected bacterial target for 6 specific regions of the corresponding foodborne pathogenic bacteria detection target sequence can further ensure the high specificity of isothermal amplification of the microfluidic chip, that is, strong specificity;

[0091] 4. The microfluidic chip and detection kit provided by the present invention can be applied to the efficient and accurate detection of foodborne pathogenic bacteria, which is of extremely important significance for improving the rapid detection ability of foodborne pathogenic bacteria in the food-related industry, especially in the pre-cut fruit and vegetable industry, and has broad market prospects and great economic and social benefits, and is suitable for large-scale popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, where:

[0093] Figure 1 is a schematic structural diagram of the microfluidic chip I provided in Embodiment 1 of the present invention; where, 1 represents a sample addition hole; 2 represents a first pre-buried area; 3 represents a treatment pool; 4 represents a first hydrophobic barrier channel; 5 represents a third pre-buried area; 6 represents a first waste liquid pool; 7 represents a quantification pool; 8 represents a second hydrophobic barrier channel; 9 represents a second pre-buried area (including a reaction pool); 10 represents a ventilation hole; 11 represents a second waste liquid pool; 12 represents a card slot positioning hole;

[0094] Figure 2 is a schematic structural diagram of the microfluidic chip II provided in Embodiment 1 of the present invention; where, 1' represents a sample addition hole; 7' represents a quantification pool; 8' represents a second hydrophobic barrier channel; 9' represents a pre-buried area (including a reaction pool); 10' represents a ventilation hole; 11' represents a waste liquid pool; 12' represents a card slot positioning hole;

[0095] Figure 3 is the amplification detection result of detecting the test sample Sm by two microfluidic chips in Embodiment 2 of the present invention; where, Figure 3 Figure A is the amplification detection result using the microfluidic chip I, Figure 3 Figure B is the amplification detection result using the microfluidic chip II;

[0096] Figure 4 is the amplification detection result of detecting the test sample Lm by two microfluidic chips in Embodiment 2 of the present invention, where, Figure 4 Figure A is the amplification detection result using the microfluidic chip I, Figure 4 Figure B is the amplification detection result using the microfluidic chip II;

[0097] Figure 5 Amplification detection results of Sm in the test sample detected in Example 3 of the present invention;

[0098] Figure 6 Amplification detection results of Sa in the test sample detected in Example 3 of the present invention;

[0099] Figure 7 Amplification detection results of Lm in the test sample detected in Example 3 of the present invention;

[0100] Figure 8 Amplification detection results of O157 in the test sample detected in Example 3 of the present invention;

[0101] Figure 9 Amplification detection results of Cro in the test sample detected in Example 3 of the present invention. Detailed implementation manners

[0102] The present invention will be further described in detail below in conjunction with specific embodiments. The present invention can be better understood according to the following embodiments. However, those skilled in the art should easily understand that the following embodiments are only descriptive and do not mean to limit the present invention to these specific embodiments. Those skilled in the art should realize that the present invention will cover all improvement schemes, alternative schemes and equivalent schemes within the scope of the claims.

[0103] Approximate terms in the specification and claims of the present application are used to modify quantities, indicating that the present invention is not limited to the specific quantity, but also includes modified parts that are close to the quantity and acceptable but will not cause changes in relevant basic functions. In the specification and claims of the present application, range limitations can be combined and / or interchanged. If not otherwise stated, these ranges also include all numerical values and sub-ranges contained therein.

[0104] In the following embodiments, the bacteria used - Salmonella enterica subsp. enterica (Sm, ATCC14028), Staphylococcus aureus (Sa, ATCC25923), Listeria monocytogenes (Lm, ATCC19115), Escherichia coli O157:H7 (O157, ATCC35150) and Cronobacter (Cronobacter sakazakii, Cro, ATCC29544) were all purchased from Beijing BioWin Biotechnology Co., Ltd.; the bacterial lysate used (composition: 1.5%wt PVP-40, 0.03%wt SDS, 2%wt Triton-100, 150 mM NaCl and 1 mM EDTA) was from Hunan Rongjian Biotechnology Co., Ltd.; the capsule material used was the aluminum foil produced by BoYu ALUMINUM Co., Ltd.; the commercial freeze-dried DNA LAMP Mix reaction solution was the MDX119 reagent produced by Meridian Co., Ltd. (an additional 8 mM Mg2+ ); The microfluidic chip supporting instrument used is the microfluidic nucleic acid analyzer BHP9609 produced by Hunan Rongjian Biotechnology Co., Ltd.

[0105] Example 1 Preparation of Microfluidic Chip

[0106] The microfluidic chip provided in this example includes a chip body and a top membrane bonded thereto. This example provides microfluidic chip I and microfluidic chip II. Figure 1 shows a schematic structural diagram of microfluidic chip I. As Figure 1 shown, microfluidic chip I is disc-shaped, with a card slot positioning hole 12 in the center, and two identical regions are symmetrically arranged along the circumference of the disc. Each region is composed of a sample loading hole 1, a first pre-embedded area 2, a processing pool 3, a first hydrophobic barrier channel 4, a third pre-embedded area 5, a first waste liquid pool 6, a quantification pool 7, a second hydrophobic barrier channel 8, a second pre-embedded area 9 (including a reaction pool), a vent hole 10, and a second waste liquid pool 11.

[0107] As Figure 2 shown, the difference between microfluidic chip II and microfluidic chip I is only that microfluidic chip II does not have a first pre-embedded area, a processing pool, a first hydrophobic barrier channel, a third pre-embedded area, and a first waste liquid pool.

[0108] In microfluidic chip I, the sample loading hole 1 is used for liquid sample loading. The sample loading liquid is a homogenized liquid sample of pre-cut fruits and vegetables to be tested. Tear off the sealing film, fit it tightly, with the side of the sealing film facing up, and stably fix the chip card slot positioning hole on the tray of the microfluidic nucleic acid analyzer BHP9609. First, centrifuge at 1500 revolutions per minute for 3 minutes at room temperature to deposit all possible microorganisms in the liquid sample to be tested into the processing pool 3; secondly, centrifuge at 2500 revolutions per minute for 5 minutes at 62 °C to rupture the liquid capsules pre-embedded in the first pre-embedded area 2, release the bacterial lysate, and flow into the processing pool 3, where bacterial lysis occurs. At the same time, the low-melting-point solid paraffin pre-embedded in the third pre-embedded area 5 melts during the heating process to seal the waste liquid containing polysaccharides and polyphenols in the liquid sample to be tested into the first waste liquid pool 6; thirdly, centrifuge at 3500 revolutions per minute for 2 minutes at 62 °C to make the liquid containing the nucleic acid components to be tested flow through the first hydrophobic barrier channel 4 into the second pre-embedded area 9. In the reaction pool of the second pre-embedded area 9, a freeze-dried mixture (a freeze-dried powder mixture containing a primer set for detecting the nucleic acid of the target bacteria, a DNA loop-mediated isothermal amplification reaction solution, and a fluorescent dye) is pre-embedded; finally, amplify and detect at 62 °C for 40 minutes, and determine the presence of the bacterial sample according to the change of the fluorescence curve. The vent hole 10 is used for air inlet and outlet to ensure smooth subsequent sample loading; the second waste liquid pool 11 is used to store the excess amplification reaction solution; the card slot positioning hole 12 is adapted to the instrument and plays a fixing role.

[0109] In microfluidic chip I and microfluidic chip II, the freeze-dried powder of the mixture pre-embedded in each reaction pool in the second pre-embedded area 9 or pre-embedded area 9' includes: ① primer sets designed according to the specific conserved gene sequences of the target bacterial genes to be detected, ② commercially available lyophilizable DNA LAMP Mix reaction solution (including Bst DNA polymerase, reaction buffer, deoxyribonucleoside triphosphates (dNTPs), and Mg 2+ (added additionally)), and ③ fluorescent dye (SYBR Green I).

[0110] The primer sets corresponding to the detected bacterial targets, the commercially available lyophilizable DNA LAMP Mix reaction solution, and the fluorescent dye are dissolved together in sterile water to obtain a freeze-dried mixture. Among them, the molar ratios between various primer pairs in each primer set are as described above, the final concentration of the commercially available lyophilizable DNA LAMP Mix reaction solution is 1X (additionally supplemented with 8 mM Mg 2+ ), and the final concentration of the fluorescent dye is 1X. The corresponding freeze-dried mixture is spotted into the corresponding target wells of the reaction pool, and then freeze-dried and top membrane bonded. After the chip bonding is completed, an aluminum bag is taken and evacuated for chip packaging to obtain microfluidic chip I and microfluidic chip II.

[0111] Example 2 Primer Design

[0112] Primer sets for the targets of Salmonella (MK017932) invA gene sequence, Staphylococcus aureus (X61307) spa gene sequence, Listeria monocytogenes (KJ504151) hylA gene sequence, Enterotoxigenic Escherichia coli O157: H7 (LC743564) rfbE gene sequence, Cronobacter (DQ000206) ompA gene, and 16S ribosomal RNA (947777) sequence in the NCBI database are designed and screened respectively. Among them, each primer set includes: outer primers B3 and F3, and inner primers FIP and BIP. The primer sets designed for the five foodborne pathogenic bacteria: Sm, Sa, Lm, O157, Cro, and the target of the bacterial endogenous gene 16S rRNA are shown in Table 2 below.

[0113] Table 2 Primer Sets

[0114]

[0115] Example 3 Comparative Experiment

[0116] Each primer set designed in Table 3 of Example 2 was pre-embedded into each reaction pool of microfluidic chip I and microfluidic chip II. Among the various pre-embedded primer sets, the molar ratio of Sm-F3 / B3:Sm-FIP / BIP was 1:8; the molar ratio of Sa-F3 / B3:Sa-FIP / BIP was 1:8; the molar ratio of Lm-F3 / B3:Lm-FIP / BIP was 1:8; the molar ratio of O157-F3 / B3:O157-FIP / BIP was 1:8; the molar ratio of Cro-F3 / B3:Cro-FIP / BIP was 1:8; the molar ratio of 16S rRNA-F3 / B3:16S rRNA-FIP / BIP was 1:8. Among them, each target primer set included a pair of outer primers and a pair of inner primers, and the molar ratio between the primers in each pair of primers in the same group was 1:1.

[0117] The purchased standard quality control strains of Sm ATCC14028 and Lm ATCC19115 were amplified and cultured. Single pure colonies were respectively picked and added to fresh-cut honeydew melons for homogenization treatment to obtain test samples containing Sm ATCC14028 (test sample Sm) and test samples containing Lm ATCC19115 (test sample Lm), respectively.

[0118] For microfluidic chip I: 200 μL of each of the two test samples was respectively added to the sample loading holes of two microfluidic chips I. The sealing film was torn off and fitted tightly. With the side with the sealing film facing up, the card slot positioning holes in the center of the chip were stably fixed on the tray of the microfluidic nucleic acid analyzer BHP9609, and the automated program was entered: First, centrifuge at 1500 revolutions per minute for 3 min at room temperature to deposit the microorganisms that may be contained in the test sample liquid to the treatment pool; Second, centrifuge at 2500 revolutions per minute for 5 min at 62 °C to rupture the liquid capsules in the first pre-embedded area, release 40 μL of bacterial lysate, and flow into the treatment pool, where bacterial lysis was carried out. At the same time, the low-melting-point solid paraffin pre-embedded in the third pre-embedded area melted during the heating process to seal the waste liquid containing polysaccharides and polyphenols into the first waste liquid pool; Third, centrifuge at 3500 revolutions per minute for 2 min at 62 °C to make the component containing nucleic acids of foodborne pathogenic bacteria (about 50 μL) flow into the reaction pool, and perform amplification detection at 62 °C for 40 min.

[0119] For the microfluidic chip II: Take 200 μL of each of the two samples to be tested in centrifuge tubes and centrifuge at 1500 revolutions per minute for 3 minutes to collect the precipitated liquid below; Add 40 μL of bacterial lysate to the precipitated liquid and vortex to mix evenly. At the same time, incubate in a metal bath at 62 °C for 5 minutes to complete bacterial lysis and release nucleic acids; Then take 50 μL each and add it to the sample loading holes of two microfluidic chips II. Tear off the sealing film, fit it tightly, with the side with the sealing film facing up, and stably fix the positioning hole of the card slot in the center of the chip on the tray of the microfluidic nucleic acid analyzer BHP9609, and enter the automated program: Centrifuge at 3500 revolutions per minute at 62 °C for 2 minutes to allow the components containing nucleic acids of foodborne pathogenic bacteria to flow into the reaction pool, and perform amplification detection at 62 °C for 40 minutes.

[0120] Determine the detection situation of the bacterial sample according to the change of the fluorescence curve. The fluorescence curve detection results are as Figures 3 to 4 shown, and the result analysis is as follows:

[0121] Figure 3 This is the amplification detection result of the two microfluidic chips for detecting the sample Sm to be tested in Example 2 of the present invention. Among them, Figure 3 Figure A is the amplification detection result of the microfluidic chip I, Figure 3 Figure B is the amplification detection result of the microfluidic chip II. From Figure 3 the fluorescence curve results, it can be seen that the negative control NEG all shows a negative curve, and both the curve Sm and the 16S rRNA target curve show a standard S shape, indicating that the Sm target has undergone isothermal amplification reactions on both microfluidic chips; at the same time, the detection times are basically the same, indicating that the sample Sm to be tested can be directly amplified and detected on both the microfluidic chip I and the microfluidic chip II, and the detection effects of the microfluidic chip I and the microfluidic chip II after multiple manual operations are equivalent.

[0122] Figure 4 This is the amplification detection result of the two microfluidic chips for detecting the sample Lm to be tested in Example 2 of the present invention. Among them, Figure 4 Figure A is the amplification detection result of the microfluidic chip I, Figure 4 Figure B is the amplification detection result of the microfluidic chip II. From Figure 4 the fluorescence curve results, it can be seen that the negative control NEG all shows a negative curve, and both the curve Lm and the 16S rRNA target curve show a standard S shape, indicating that the Lm target has undergone isothermal amplification reactions on both microfluidic chips; at the same time, the detection times are basically the same, indicating that the sample Lm to be tested can be directly amplified and detected on both the microfluidic chip I and the microfluidic chip II, and the detection effects of the microfluidic chip I and the microfluidic chip II after multiple manual operations are equivalent.

[0123] Example 4 Detection experiment of fresh-cut fruit samples

[0124] The detection experiment of this example was carried out using the microfluidic chip I. Each primer set designed in Table 3 of Example 2 was pre-embedded into each reaction pool of the microfluidic chip I. Among various pre-embedded primer sets, the molar ratio of Sm-F3 / B3:Sm-FIP / BIP was 1:8; the molar ratio of Sa-F3 / B3:Sa-FIP / BIP was 1:8; the molar ratio of Lm-F3 / B3:Lm-FIP / BIP was 1:8; the molar ratio of O157-F3 / B3:O157-FIP / BIP was 1:8; the molar ratio of Cro-F3 / B3:Cro-FIP / BIP was 1:8; the molar ratio of 16S rRNA-F3 / B3:16S rRNA-FIP / BIP was 1:8. Among them, each target primer set included a pair of outer primers and a pair of inner primers, and the molar ratio between primers in each pair of primers in the same group was 1:1.

[0125] Five standard quality control strains of foodborne pathogenic bacteria, namely Sm ATCC14028, Sa ATCC25923, Lm ATCC19115, O157 ATCC35150 and Cro ATCC29544, were purchased and amplified. Single pure colonies were respectively picked and added to fresh-cut honeydew melons and then homogenized to obtain test samples containing Sm ATCC14028 (test sample Sm), test samples containing Sa ATCC25923 (test sample Sa), test samples containing Lm ATCC19115 (test sample Lm), test samples containing O157 ATCC35150 (test sample O157) and test samples containing Cro ATCC29544 (test sample Cro).

[0126] 200 μL of each of the five test samples was respectively added to the sample loading holes of five microfluidic chips I. The sealing film was torn off, and they were closely attached. With the side of the sealing film facing up, the card slot positioning holes in the center of the chip were stably fixed on the tray of the microfluidic nucleic acid analyzer BHP9609, and the automated program was entered: First, centrifuge at 1500 revolutions per minute for 3 min at room temperature to deposit the microorganisms that might be contained in the test sample liquid to the processing pool; second, centrifuge at 2500 revolutions per minute for 5 min at 62 °C to rupture the liquid capsules pre-embedded in the first pre-embedded area, release 40 μL of bacterial lysate, and flow into the processing pool, where bacterial lysis was carried out. At the same time, the low-melting-point solid paraffin pre-embedded in the third pre-embedded area melted during the heating process to seal the waste liquid containing polysaccharides and polyphenols into the first waste liquid pool; third, centrifuge at 3500 revolutions per minute for 2 min at 62 °C to make the components containing nucleic acids of foodborne pathogenic bacteria flow into the reaction pool, and perform amplification detection at 62 °C for 40 min.

[0127] The detection situation of foodborne pathogenic bacteria in the test sample was determined according to the change of the fluorescence curve. The fluorescence curve detection results are asFigures 5 to 9 As shown, the result analysis is as follows:

[0128] Figure 5 This is the amplification detection result of detecting the sample Sm to be tested in Example 3 of the present invention. From Figure 5 the fluorescence curve results, it can be seen that the negative control NEG is a negative curve, the curves of Sm and 16S rRNA detection targets are in a standard S shape, and no peaks appear for other targets, indicating that only the Sm and 16S rRNA detection targets have undergone isothermal amplification reactions. The microfluidic chip I of the present invention can achieve direct amplification detection of fresh-cut melon samples containing Sm.

[0129] Figure 6 This is the amplification detection result of detecting the sample Sa to be tested in Example 3 of the present invention. From Figure 6 the fluorescence curve results, it can be seen that the negative control NEG is a negative curve, the curves of Sa and 16S rRNA detection targets are in a standard S shape, and no peaks appear for other targets, indicating that only the Sa and 16S rRNA detection targets have undergone isothermal amplification reactions. The microfluidic chip I of the present invention can achieve direct amplification detection of fresh-cut melon samples containing Sa.

[0130] Figure 7 This is the amplification detection result of detecting the sample Lm to be tested in Example 3 of the present invention. From Figure 7 the fluorescence curve results, it can be seen that the negative control NEG is a negative curve, the curves of Lm and 16S rRNA detection targets are in a standard S shape, and no peaks appear for other targets, indicating that only the Lm and 16S rRNA detection targets have undergone isothermal amplification reactions. The microfluidic chip I of the present invention can achieve direct amplification detection of fresh-cut melon samples containing Lm.

[0131] Figure 8 This is the amplification detection result of detecting the sample O157 to be tested in Example 3 of the present invention. From Figure 8 the fluorescence curve results, it can be seen that the negative control NEG is a negative curve, the curves of O157 and 16S rRNA detection targets are in a standard S shape, and no peaks appear for other targets, indicating that only the O157 and 16S rRNA detection targets have undergone isothermal amplification reactions. The microfluidic chip I of the present invention can achieve direct amplification detection of fresh-cut melon samples containing O157.

[0132] Figure 9 This is the amplification detection result of detecting the sample Cro to be tested in Example 3 of the present invention. From Figure 9From the fluorescence curve results, it can be seen that the negative control NEG shows a negative curve, the curves of Cro and the 16S rRNA detection target show a standard S shape, and no peaks appear for other targets, indicating that only the Cro and 16S rRNA detection targets have undergone isothermal amplification reactions. The microfluidic chip I of the present invention can achieve direct amplification detection of fresh-cut melon samples containing Cro.

[0133] From the above experimental results, it can be known that the microfluidic chip of the present invention is based on the LAMP method and microfluidic technology, and through a special structural design, only the sample to be tested needs to be homogenized, and subsequent nucleic acid component collection, lysis and detection can all be completed within the microfluidic chip, greatly reducing manual operations and reducing aerosol contamination. It can be applied to the direct amplification detection of foodborne pathogenic bacteria in fresh-cut fruit and vegetable samples, and the detection effect is equivalent to that after multiple manual operation steps; it has the characteristics of fast, efficient and simple, and at the same time has the advantage of detecting multiple target genes with a single sample addition. The microfluidic chip of the present invention is easy to operate. Only the homogenized fruit sample solution needs to be simply mixed and then added to the sample and sealed. The whole system is convenient and controllable to operate; it can also detect multiple samples at the same time. The whole detection process can be completed within 50 minutes, and only a single sample addition is required for each area to simultaneously detect five foodborne pathogenic bacteria, and the specific presence of foodborne pathogenic bacteria in fresh-cut fruit and vegetable samples can be determined quickly and efficiently.

[0134] Although various aspects and embodiments of the present invention are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and not for limiting purposes. The scope and gist of the present invention are determined only by the appended claims.

Claims

1. A microfluidic chip for detecting foodborne pathogens, characterized in that: The microfluidic chip includes at least one detection area, and the detection area includes: A processing tank, which is used to collect and lyse bacterial cells in the liquid sample to be tested; A first pre-embedded area, in which components for lysing bacterial cells are pre-embedded; A second pre-embedded area, in which the reaction pool has pre-embedded components for detecting bacterial nucleic acid by loop-mediated isothermal amplification; Wherein, the treatment pool is connected with the reaction pools in the first pre-buried area and the second pre-buried area respectively via fluid channels.

2. The microfluidic chip according to claim 1, characterized in that: The detection area also includes: The first waste liquid tank is connected with the treatment tank via a fluid channel.

3. The microfluidic chip according to claim 1, characterized in that: The detection area also includes: The third pre-buried area has a medium pre-buried therein for sealing the first waste liquid pool.

4. The microfluidic chip according to claim 1, characterized in that: The treatment tank is fluidly connected to the reaction tank in the second pre-buried area via a hydrophobic barrier channel.

5. The microfluidic chip according to claim 4, characterized in that: The width of the hydrophobic barrier channel is less than 100 microns.

6. The microfluidic chip according to any one of claims 1 to 4, characterized in that: The second pre-embedded area includes at least two reaction pools, and components used to detect different types of bacteria through loop-mediated isothermal amplification are pre-embedded in different reaction pools.

7. The microfluidic chip according to any one of claims 1 to 4, characterized in that: The detection area also includes: A sample addition hole, which is connected to the processing pool via a fluid channel; A quantitative pool, which is arranged between the treatment pool and the reaction pool in the second pre-buried area, and is respectively in fluid communication with the treatment pool via a first hydrophobic barrier channel and in fluid communication with the reaction pool in the second pre-buried area via a second hydrophobic barrier channel; A second waste liquid pool is connected to the reaction pool in the second pre-buried area via a fluid channel; and / or Breathing holes.

8. The microfluidic chip according to any one of claims 1 to 4, characterized in that: The components for detecting bacterial nucleic acids by loop-mediated isothermal amplification include a primer set for detecting foodborne pathogens, a DNA loop-mediated isothermal amplification reaction solution, and a freeze-dried mixture of a fluorescent dye, wherein the DNA loop-mediated isothermal amplification reaction solution contains DNA polymerase, deoxyribonucleoside triphosphates, a magnesium ion solution, and a reaction buffer.

9. The microfluidic chip according to claim 8, characterized in that: The primer set for detecting foodborne pathogenic bacteria includes one or more primer sets selected from the following: a primer set for detecting Salmonella, a primer set for detecting Staphylococcus aureus, a primer set for detecting Listeria monocytogenes, a primer set for detecting diarrhea-causing Escherichia coli, a primer set for detecting Cronobacter and a primer set for detecting bacterial 16S rRNA gene.

10. The microfluidic chip according to claim 9, characterized in that: The primer set for detecting Salmonella includes: The outer primer pair Sm-F3 and Sm-B3, whose nucleotide sequences are shown in SEQ ID NOs. 1 and 2, respectively; and The inner primer pair Sm-FIP and Sm-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 3 and 4, respectively; The primer set for detecting Staphylococcus aureus includes: The outer primer pair Sa-F3 and Sa-B3, whose nucleotide sequences are shown in SEQ ID NOs. 5 and 6, respectively; and The inner primer pair Sa-FIP and Sa-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 7 and 8, respectively; The primer set for detecting Listeria monocytogenes includes: The outer primer pair Lm-F3 and Lm-B3, whose nucleotide sequences are shown in SEQ ID NOs. 9 and 10, respectively; and The inner primer pair Lm-FIP and Lm-BIP, the nucleotide sequences of which are shown in SEQ ID NOs. 11 and 12, respectively; The primer set for detecting diarrhea-causing Escherichia coli comprises: The outer primer pair O157-F3 and O157-B3, whose nucleotide sequences are shown in SEQ ID NOs. 13 and 14, respectively; and The inner primer pair O157-FIP and O157-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 15 and 16, respectively; The primer set for detecting Cronobacter comprises: The outer primer pair Cro-F3 and Cro-B3, whose nucleotide sequences are shown in SEQ ID NOs. 17 and 18, respectively; and The inner primer pair Cro-FIP and Cro-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 19 and 20, respectively; The primer set for detecting bacterial 16S rRNA includes: The outer primer pair 16S rRNA-F3 and 16S rRNA-B3, whose nucleotide sequences are shown in SEQ ID NOs. 21 and 22, respectively; and The nucleotide sequences of the inner primer pair 16S rRNA-FIP and 16S rRNA-BIP are shown in SEQ ID NOs. 23 and 24, respectively.

11. A method for detecting foodborne pathogens, characterized in that: The method comprises using the microfluidic chip according to any one of claims 1 to 10 to perform nucleic acid detection of foodborne pathogens.

12. The method according to claim 11, characterized in that The method comprises using the microfluidic chip according to any one of claims 1 to 10 to sequentially collect and lyse bacteria and perform loop-mediated isothermal amplification reaction on the homogenized liquid sample to be tested, and determining the presence of bacteria in the sample to be tested based on the generated fluorescence curve.

13. The method according to claim 11 or 12, characterized in that: The method comprises the following steps: (1) adding the homogenized liquid sample to be tested to the sample loading hole of the microfluidic chip, and centrifuging at 1000-1800 rpm, for example, 1500 rpm, so that the bacterial cells in the liquid sample to be tested are collected in the processing pool; (2) centrifuging and heating at 2000-2800 rpm, for example, 2500 rpm, to release the components used for lysing bacterial cells pre-embedded in the first pre-embedded area, lysing the bacterial cells in the liquid sample to be tested in the treatment tank, and at the same time, the low-melting-point solid paraffin pre-embedded in the third pre-embedded area seals the waste liquid generated after the liquid sample to be tested is collected and treated with bacteria into the first waste liquid tank; (3) Centrifuging at 3000-4000 rpm, for example, 3500 rpm, allows the bacterial nucleic acid in the liquid sample to enter the reaction pool in the second pre-embedded area, and detecting the bacterial nucleic acid by loop-mediated isothermal amplification.

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