Microfluidic device for detecting salmonella and detection method

Abstract

The invention relates to the technical field of microbiological detection, and particularly discloses a microfluidic device for detecting salmonella and a detection method.The microfluidic device comprises a top cover, a functional plate and a heating base which are detachably connected in sequence from top to bottom; an observation window, a first sample adding opening and a second sample adding opening are formed in the top cover; a detection bin is formed in a position, corresponding to the observation window, on the function plate; an amplification bin communicated with the second sample adding opening is formed in the functional plate; a buffer bin communicated with the first sample adding opening is formed in the functional plate; the amplification bin is communicated with the buffer bin, the buffer bin is communicated with the detection bin, and a space for placing a test strip is reserved in the detection bin; a ventilation channel is further formed in the function plate and communicates with the amplification bin. The micro-fluidic device provided by the invention is simple in structure, convenient to carry, easy to interpret a detection result and simple in operation mode, and meanwhile, in combination with the design of the micro-fluidic device, the nucleic acid detection does not need additional equipment and complicated manual intervention.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a microfluidic device and a detection method for detecting Salmonella. Background Art

[0002] Foodborne pathogens are one of the main factors causing foodborne diseases. Salmonella is a pathogen that is widely distributed in nature. It can survive for several months in water, meat and dairy products, posing a serious threat to human and animal health.

[0003] Recombinase polymerase amplification is a new type of nucleic acid constant temperature amplification technology developed by the British company TwistDx Inc in 2006. It can not only detect DNA but also RNA. The entire reaction relies on three key enzymes: polymerase, recombinase, and single-stranded binding protein. Compared with other constant temperature amplification technologies, it reacts at a lower operating temperature of 37°C to 42°C and can complete effective amplification in about 30 minutes.

[0004] However, the existing rapid detection technology requires professional equipment to view the results. After the PCR instrument and agarose gel electrophoresis instrument RPA amplification reaction are completed, the lid needs to be opened to take out the amplification product for transfer and detection. After opening the lid, the target gene is easy to fly into the air, so that the air is filled with the target gene, and it is easy to cause false positives when the nucleic acid is tested again. Aerosol pollution causes false positives.

[0005] Therefore, it is necessary to develop a simple and portable RPA detection device. Summary of the invention

[0006] In order to develop a simple and portable RPA detection device, the present invention provides a microfluidic device and a detection method for detecting Salmonella. The microfluidic device for detecting Salmonella provided by the present invention has a simple structure, is easy to carry, and the detection results are easy to interpret and the operation method is simple. At the same time, the design of the microfluidic device makes nucleic acid detection without additional equipment and complex manual intervention.

[0007] The present invention provides a microfluidic device for detecting Salmonella, characterized in that it comprises a top cover, a functional plate and a heating base which are detachably connected in sequence from top to bottom; The top cover is provided with a transparent observation window, a first centrifugal hole, a first sample adding port and a second sample adding port; A detection chamber is provided on the functional board at a position corresponding to the observation window; an amplification chamber connected to the second sample loading port is provided on the functional board; and a buffer chamber connected to the first sample loading port is provided on the functional board; The amplification chamber is reserved with a space for filling a freeze-dried reagent; the freeze-dried reagent contains a Salmonella-specific primer; The amplification chamber is connected to the buffer chamber, the buffer chamber is connected to the detection chamber, and a space for placing a test strip is reserved in the detection chamber; The functional plate is also provided with a ventilation channel, one end of which is connected to the amplification chamber, and the other end of which is connected to the detection chamber; A second centrifugal hole is provided on the functional plate corresponding to the position of the first centrifugal hole, and the first centrifugal hole and the second centrifugal hole are connected; The heating base is used to be connected to a power source.

[0008] The present invention provides a microfluidic device consisting only of a top cover, a functional board and a heating base, wherein the top cover is provided with an observation window, a second sample addition port and a first sample addition port, and the functional board is provided with a detection chamber, an amplification chamber and a buffer chamber correspondingly. The microfluidic device has a simple structure, is compact and portable, and can be directly used for outdoor RPA reactions. It only needs to add the detection system to the microfluidic device to accurately detect whether the sample contains Salmonella. The operation is simple, and the detection is performed immediately after the amplification reaction of the present invention, without taking out the amplification product, which can avoid the target gene from flying and avoid aerosol contamination and false positive phenomenon.

[0009] Furthermore, the amplification chamber is connected to the buffer chamber via a first communication channel, and the buffer chamber is connected to the detection chamber via a second communication channel, and the pipe diameters of the first communication channel and the second communication channel are both 0.02 mm to 2 mm. Liquid will not flow freely through the pipe, so that the liquid in the amplification chamber will not flow out of the amplification chamber at will with the movement of the human body.

[0010] Furthermore, a first sealing cover is provided on the movable sleeve of the first sample adding port, and a second sealing cover is provided on the movable sleeve of the second sample adding port.

[0011] Furthermore, a heating plate is provided on the heating base.

[0012] Furthermore, the amplification chamber is connected to the buffer chamber via a first connecting channel, and the buffer chamber is connected to the detection chamber via a second connecting channel.

[0013] Furthermore, the test strip is a colloidal gold immunochromatography test strip.

[0014] Furthermore, the observation window is a partition made of transparent material, which separates the detection chamber from the outside world and the reaction situation in the detection chamber can be observed through the observation window.

[0015] The present invention provides a method for detecting Salmonella, wherein the method for detecting Salmonella is based on the microfluidic device for detecting Salmonella and comprises the following steps: The test strip is prepared according to the preparation steps of the colloidal gold immunochromatographic test strip; the prepared test strip includes a detection line T line and a quality control line C line, wherein the T line is loaded with streptavidin and the C line is loaded with goat anti-mouse antibody; Preparation for testing: a freeze-dried reagent containing specific primers for detecting Salmonella is added into the amplification chamber, the freeze-dried reagent is used to store the reagents required for the RPA amplification reaction, a PBS buffer for stabilizing the pH value of the reaction system is added into the buffer chamber through the first sample addition port, and a test strip is placed in the detection chamber; Add the lysate of the sample to be tested to the second sample injection port, and the sample reaches the amplification chamber through the second sample injection port, seal the second sample injection port, heat the amplification chamber to 37° C. to 42° C. using the heating base, and maintain heating for 30 min to 40 min to perform RPA amplification reaction to obtain an amplification product; The fixing belt is passed through the first centrifugal hole and the second centrifugal hole for fixing, and then the amplification product in the amplification chamber is transferred to the buffer chamber for dilution by centrifugal force, and the diluted amplification product flows into the detection chamber to contact the test strip and be detected; then the test strip is observed through the observation window and judged, and the judgment standard is: the T line and the C line are both positive, indicating that Salmonella is detected; the T line is not colored and the C line is negative, indicating that Salmonella is not detected.

[0016] Furthermore, the specific primer pair for detecting Salmonella includes an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2.

[0017] Furthermore, the upstream primer is 5' labeled with biotin, and the downstream primer is 5' labeled with 6-aminofluorescein.

[0018] Furthermore, the lyophilized reagent contains the following components at final concentrations: 520 ng / μL to 725 ng / μL recombinase, 100 ng / μL to 200 ng / μL single-stranded DNA binding protein, 350 ng / μL to 450 ng / μL DNA polymerase, 200 nM to 400 nM upstream primer, 200 nM to 400 nM downstream primer, 4% to 5% polyethylene glycol by mass, 18 mM to 20 mM tris(hydroxymethyl)aminomethane hydrochloride, 180 mM to 200 mM sodium acetate, 8 mM to 10 mM dithiothreitol, 8 mM to 10 mM adenosine triphosphate, 90 mM to 100 mM creatine phosphate disodium salt, 100 ng / μL to 120 ng / μL phosphatase, 480 uM~500uM deoxyribonucleoside triphosphate, mass fraction 9%~10% trehalose, 40 ng / μL~50 ng / μL mannitol.

[0019] Furthermore, the sample to be tested is lysed with a lysing solution to obtain the sample lysate to be tested.

[0020] Furthermore, 500 μL of the lysate includes a final concentration of 50 mM to 100 mM tris(hydroxymethyl)aminomethane hydrochloride, a final concentration of 50 mM to 80 mM ethylenediaminetetraacetic acid, a final concentration of 2 wt% to 5 wt% Tween, a final concentration of 0.5 mM to 2 mM guanidine hydrochloride, a final concentration of 0.5 mM to 2 mM guanidine isothiocyanate, a final concentration of 0.5 M to 1 M sodium chloride, a final concentration of 2 wt% to wt5% sodium dodecyl sulfate and a final concentration of 23 wt% to 25 wt% sodium dodecyl sarcosine, 14 mM to 18 mM magnesium acetate, and the solvent is deionized water.

[0021] The present invention also provides a primer pair for detecting Salmonella, comprising an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2.

[0022] The present invention also provides an application of a primer pair for detecting Salmonella in preparing a reagent for identifying Salmonella. The primer pair for detecting Salmonella comprises an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2. The Salmonella is Salmonella enterica.

[0023] Furthermore, the reagent can specifically distinguish Salmonella from Salmonella, Staphylococcus aureus, Staphylococcus waldenii, Micrococcus luteus, Escherichia coli and Listeria monocytogenes.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a microfluidic device for detecting Salmonella, which is composed of a top cover, a functional board and a heating base, wherein the top cover is provided with an observation window, a second sample adding port and a first sample adding port, and the functional board is provided with a detection chamber, an amplification chamber and a buffer chamber. The microfluidic device has a simple structure and is portable, can be detected without opening the cover, can avoid aerosol contamination, and avoids causing false positive phenomenon.

[0025] The present invention also provides a method for detecting Salmonella, which adopts a method combining rapid constant temperature amplification technology and immunochromatographic test strips, designs primers for Salmonella-specific gene sequences, and modifies the primers so that the amplified product can be captured and colored at the T line when it is on the test strip; the whole process is simple to operate and the results are visualized, and it does not rely on detection equipment, environment and personnel, and the results can be easily detected and read. The primer pair Sal-F and Sal-R in the present invention are highly specific and sensitive to Salmonella, and the primer pair can specifically identify Salmonella from Salmonella, Staphylococcus aureus, Staphylococcus waldenii, Micrococcus luteus, Escherichia coli and Listeria monocytogenes.

[0026] The method for detecting Salmonella using a microfluidic device of the present invention only relies on the microfluidic device to independently complete the entire Salmonella detection process, does not require additional equipment and complex manual intervention, and avoids aerosol pollution during the operation process. The overall detection time is short, the detection results are easy to interpret, the operation method is simple, and it is suitable for home self-testing of Salmonella. The detection method has high efficiency, high specificity and high sensitivity, and has a high detection accuracy rate.

[0027] The microfluidic device for detecting Salmonella provided by the present invention optimizes the design of the circulation pipeline, that is, the pipeline diameters of the first connecting channel and the second connecting channel 12 are designed to be 1 mm, so that the pipeline is thinner, avoiding the use of liquid valves, and the liquid will not flow freely through the pipeline, so that the liquid in the amplification chamber will not flow out of the amplification chamber at will with the movement of the human body; at the same time, the second connecting channel between the detection chamber and the buffer chamber is set to be tilted downward near one end of the detection chamber, so that the amplification product will not flow back; through the action of the centrifugal hole rope, the amplification product can be made to flow in a unidirectional controlled manner, thereby realizing the rapid detection of Salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 A schematic structural diagram of a microfluidic device for detecting Salmonella provided by the present invention.

[0030] Figure 2 To use Figure 1 The test results of the device testing samples are meat paste No. 1 and meat paste No. 2.

[0031] Figure 3 To use Figure 1Schematic diagram of the test results of the device detecting samples of different concentrations; in the figure, numbers 1 to 5 from left to right respectively indicate that the concentrations of the detected samples are 28.4 ng / μL, 0.284 ng / μL, 2.84 pg / μL, 0.284 pg / μL and 28.4 fg / μL.

[0032] Figure 4 A schematic diagram of specific detection results of the method for detecting Salmonella provided by the present invention; in the figure, numbers 1 to 6 from left to right respectively represent that the test samples are Salmonella enterica CGMCC 1.755, Staphylococcus aureus ZFM1CGMCC 1.184, Staphylococcus waldenii ZFM1 ACCC 06483, Micrococcus luteus ZFM1 CICC 10209, Escherichia coli O104 and Listeria monocytogenes J4045 CGMCC 1.9144.

[0033] Figure 5 The specificity of detection of Salmonella by Example 1 of the present invention and Comparative Examples 1 to 2 is compared.

[0034] Explanation of the figure numbers: 1-amplification chamber, 2-buffer chamber, 3-first sealing cover, 4-detection chamber, 5-second sealing cover, 6-second centrifugal hole, 7-first centrifugal hole, 8-ventilation channel, 9-first sample addition port, 10-second sample addition port, 11-observation window, 12-second connecting channel, 13-first connecting channel. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0036] Example 1: A microfluidic device and detection method for detecting Salmonella.

[0037] 1. Microfluidic device for detection of Salmonella like Figure 1 As shown, this embodiment provides a microfluidic device for detecting Salmonella, comprising a top cover, a functional plate and a heating base which are detachably connected in sequence from top to bottom; The top cover is provided with a transparent observation window 11, a first centrifugal hole 7, a first sample addition port 9 and a second sample addition port 10; the first sample addition port 9 is threadedly connected to a first sealing cover 3, and the second sample addition port 10 is threadedly connected to a second sealing cover 5; the observation window 11 is a plastic partition; A detection chamber 4 is provided on the functional board at a position corresponding to the observation window 11; an amplification chamber 1 is provided on the functional board corresponding to and connected with the second sample loading port 10; and a buffer chamber 2 is provided on the functional board corresponding to and connected with the first sample loading port 9; The amplification chamber 1 is connected to the buffer chamber 2 through a first connecting channel 13, and the buffer chamber 2 is connected to the detection chamber 4 through a second connecting channel 12. A colloidal gold immunochromatography test strip is placed in the detection chamber 4. The inner diameters of the first connecting channel 13 and the second connecting channel 12 are both 1 mm. The first connecting channel 13 is tilted downward near the buffer chamber 24, and the second connecting channel 12 is tilted downward near the detection chamber 4, so that the liquid can flow to the detection chamber 4 more easily to prevent backflow.

[0038] The functional plate is also provided with a ventilation channel 8, one end of which is connected to the amplification chamber 1, and the other end of which is connected to the detection chamber 4, so that the liquid can flow smoothly; A second centrifugal hole 6 is provided at a position corresponding to the first centrifugal hole 7 on the functional plate, and the first centrifugal hole 7 and the second centrifugal hole 6 are connected. A small rope is passed through the first centrifugal hole 7 and the second centrifugal hole 6, and centrifugal force is obtained by swinging. The heating base is provided with a silicone rubber heating plate and is electrically connected to a power source through a power cord. The heating base is placed directly below the amplification chamber and is used for heating the amplification reaction at a heating temperature of 42°C.

[0039] The silicone rubber heating plate is provided by Yancheng Huakai Electric Heating Appliance, with the following parameters: size 50mm x 50mm, heating temperature 40 degrees, voltage 5V.

[0040] 2. Method for detecting Salmonella using the above-mentioned microfluidic device 1. Preparation of colloidal gold immunochromatographic test strips The colloidal gold immunochromatographic test strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane, an absorption pad and a bottom plate; the 6-aminofluorescein antibody is combined with the colloidal gold, potassium carbonate is added to adjust the pH, and after oscillation for 1 hour, 20 μL of bovine serum albumin is added, and after oscillation for 1 minute, the supernatant is removed and the solution is dissolved for later use; the conjugate pad is placed on a conveying device, and the prepared colloidal gold is sprayed on the conjugate pad with a gold sprayer, and the conjugate pad is sent to a drying oven and dried at 37°C for 12 hours; a test line and an automatic control line are coated on the NC membrane; the test line T is coated with 1.5 mg / mL streptavidin, and the automatic control line C is coated with 1.2 mg / mL goat anti-mouse polyclonal antibody; the labeled conjugate pad and NC membrane are placed in an oven and dried at 37°C for 12 hours. Then, the various parts are assembled on the PVC bottom plate according to conventional operations and cut to obtain the colloidal gold immunochromatographic test strip.

[0041] 2. Preparation of lyophilized reagents The reaction solution containing a final concentration of 600 ng / μL recombinase, 200 ng / μL single-stranded DNA binding protein, 400 ng / μL DNA polymerase, 200-400 nM upstream primer, 200-400 nM downstream primer, 5% polyethylene glycol, 20 mM tris(hydroxymethyl)aminomethane hydrochloride, 200 mM sodium acetate, 10 mM dithiothreitol, 10 mM adenosine triphosphate, 100 mM creatine phosphate disodium salt, 120 ng / μL phosphatase, 500 uM deoxyribonucleoside triphosphate, 10% trehalose and 50 ng / μL mannitol was pre-frozen at -80°C for 1 h to obtain a pre-frozen reagent; the pre-frozen reagent was first dried at -35°C for 4 h, and then dried at 15°C for 1.5 h to obtain a lyophilized reagent. The upstream primer is Sal-F shown in SEQ ID NO.1, and the downstream primer is Sal-R primer shown in SEQ ID NO.2.

[0042] SEQ ID NO. 1: ATTTCTATGTTCGTCATTCCATTACCTACC.

[0043] SEQ ID NO. 2: CTGTCAATGTAGAACGACCCCATAAACACC.

[0044] The upstream primer is 5' labeled with biotin, and the downstream primer is 5' labeled with 6-aminofluorescein. The size of the amplified target product is 107 bp.

[0045] 3. Detection steps Processing of the sample to be tested: lysing the sample to be tested with a lysing solution to obtain a lysing solution of the sample to be tested for standby use; 500 μL of the lysate includes the following components at final concentrations: 100 mM Tris-HCl, 80 mM EDTA, 5 wt% Tween, 2 mM guanidine hydrochloride, 2 mM guanidine isothiocyanate, 1 M NaCl, 5 wt% sodium dodecyl sulfate and 25 wt% sodium dodecyl sarcosinate, and the solvent is deionized water.

[0046] The top cover is connected to the function plate, and the function plate is placed on the heating base.

[0047] Detection preparation: add freeze-dried reagent into amplification chamber 1, add 200 μL PBS buffer into buffer chamber 2, and place colloidal gold immunochromatography test strip into the detection chamber 4; 50 μL of the lysate of the sample to be tested is added dropwise into the second sample injection port 10, and the sample to be tested reaches the amplification chamber 1 through the second sample injection port 10, and the second sample injection port 10 is sealed, and the heating base is connected to a 5V power supply, and the amplification chamber 1 is heated to 40° C. by the heating base, and the heating is maintained for 40 min to perform RPA amplification reaction to obtain an amplification product; A small rope is passed through the first centrifugal hole 7 and the second centrifugal hole 6, and a centrifugal force is obtained by swinging, so that the amplification product in the amplification chamber 1 is transferred to the buffer chamber 2 along the connecting channel for dilution, and the diluted amplification product then flows into the detection chamber 4 to contact with the colloidal gold immunochromatographic test strip and be detected; After 15 minutes, the colloidal gold immunochromatographic test strip is observed through the observation window 11 and judged according to the following criteria: if both the T line and the C line are colored, it is positive; if the T line is not colored and the C line is colored, it is negative.

[0048] Feasibility verification: Prepare 2 chicken samples without Salmonella, numbered 1 and 2, add 1mL PBS to sample 1, and add 1mL Salmonella solution to sample 2; then, add 9mL PBS to samples 1 and 2, mix well, transfer the liquid to a 15mL centrifuge tube, centrifuge at 1000rpm for 6min to remove larger impurities, transfer the supernatant to a new 15mL centrifuge tube, centrifuge at 6000rpm for 6min to collect the precipitate, and record it as meat paste No. 1 and meat paste No. 2. Salmonella solution is composed of Salmonella enterica Salmonella enterica The CGMCC 1.755 strain was purified by streaking, and a single colony was picked and inoculated into LB liquid culture medium and cultured at 220 rpm and 37° C. for 16 h to obtain a Salmonella liquid.

[0049] Use the uncontaminated meat paste No. 1 and the meat paste No. 2 contaminated with Salmonella as the test samples, and perform the test according to the above steps. After the test, take out the colloidal gold immunochromatographic test strips and take photos to record the test results.

[0050] The results are as follows Figure 2 As shown, the test results of No. 2 meat paste contaminated with Salmonella showed that both the T line and the C line were positive; the test results of No. 1 meat paste that was not contaminated showed that the T line did not show any color and the C line was negative.

[0051] 3. Sensitivity and specificity tests of the above-mentioned methods for detecting Salmonella 1. Sensitivity analysis experiment: The prepared Salmonella solution was diluted by 10-fold concentration gradient to obtain Salmonella solutions with concentrations of 28.4 ng / μL, 0.284 ng / μL, 2.84 pg / μL, 0.284 pg / μL, 28.4 fg / μL and 2.84 fg / μL, respectively, and genomic DNA was extracted as DNA template for LFD-RPA sensitivity analysis experiment. The Salmonella is Salmonella enterica CGMCC 1.755.

[0052] Preparation of RPA reaction system: Sal-F shown in SEQ ID NO.1 and Sal-R shown in SEQ ID NO.2 were used as amplification primers. The upstream primer was 5' labeled with biotin, and the downstream primer was 5' labeled with 6-aminofluorescein. The size of the amplified target product was 107 bp.

[0053] SEQ ID NO. 1: ATTTCTATGTTCGTCATTCCATTACCTACC.

[0054] SEQ ID NO. 2: CTGTCAATGTAGAACGACCCCATAAACACC.

[0055] Each 50 μL RPA reaction system included 29.4 μL A buffer, 2 μL upstream primer Sal-F, 2 μL downstream primer Sal-R, 5 μL DNA template and 9.1 μL ddHO. 2 O, 2.5 μL B buffer.

[0056] A buffer and B buffer are from the Amp Future DNA Constant Temperature Rapid Amplification Kit (Basic Type), the product number of which is: WLB8201KIT.

[0057] The results are as follows Figure 3 As shown, the lowest bacterial liquid concentration detected by the microfluidic device and detection method of the present invention is 0.284 pg / μL, indicating that the method of the present invention has high sensitivity.

[0058] 2. Specificity experiment: The specificity of the LFD-RPA method was analyzed by detecting Salmonella enterica CGMCC 1.755, Staphylococcus aureus ZFM1 CGMCC1.184, Staphylococcus varroa ZFM1 ACCC 06483, Micrococcus luteus ZFM1 CICC 10209, Escherichia coli O104 with a deposit number of ATCCBAA-2326, and Listeria monocytogenes J4045 CGMCC 1.9144.

[0059] DNA extracted from Salmonella enterica CGMCC 1.755, Staphylococcus aureus ZFM1 CGMCC 1.184, Staphylococcus waldenii ZFM1 ACCC 06483, Micrococcus luteus ZFM1 CICC 10209, Escherichia coli O104 with a deposit number of ATCC BAA-2326, and Listeria monocytogenes J4045 CGMCC 1.9144 were used as DNA templates and detected according to the above sensitivity analysis detection steps.

[0060] Each 50 μL RPA reaction system included 29.4 μL A buffer, 2 μL upstream primer Sal-F, 2 μL downstream primer Sal-R, 5 μL DNA template and 9.1 μL ddHO. 2 O, 2.5 μL B buffer.

[0061] The results are as follows Figure 4 The test results of Salmonella enterica CGMCC 1.755, Staphylococcus aureus ZFM1 CGMCC 1.184, Staphylococcus varroa ZFM1 ACCC 06483, Micrococcus luteus ZFM1 CICC 10209, Escherichia coli O104 and Listeria monocytogenes J4045 CGMCC 1.9144 are shown in the figure from left to right. This shows that the invention of the present invention can specifically detect Salmonella, but cannot detect other strains. Therefore, the detection primer of the present invention has high specificity and the detection method is accurate.

[0062] Comparative Example 1: A method for detecting Salmonella.

[0063] The microfluidic device of Example 1 was used to detect Salmonella, and the DNA extracted from the 0.284 ng / μL Salmonella solution in Example 1 was used as a DNA template, and the RPA reaction system was the same. The difference was that the upstream primer in the RPA reaction system was shown in SEQ ID NO.3, and the downstream primer was shown in SEQ ID NO.4.

[0064] SEQ ID NO. 3: ATTTTAAATTCCGTGAAGCAAAACGTAGCGC.

[0065] SEQ ID NO.4: GATCGCCAATCAGTCCTAACGACGACCCTTC.

[0066] Comparative Example 2: A method for detecting Salmonella.

[0067] The microfluidic device of Example 1 was used to detect Salmonella, and the DNA extracted from the 0.284 ng / μL Salmonella solution in Example 1 was used as a DNA template, and the RPA reaction system was the same. The difference was that the upstream primer in the RPA reaction system was shown as SEQ ID NO.5, and the downstream primer was shown as SEQ ID NO.6.

[0068] SEQ ID NO.5: GTCATTCCATTACCTACCTATCTGGTTGATTTCC.

[0069] SEQ ID NO. 6: GCATCGGCTTCAATCAAGATAAGACGACTGGT.

[0070] The methods of Example 1, Comparative Example 1 and Comparative Example 2 were used to detect the Salmonella in the present invention. The results are as follows: Figure 5 As shown, only the T line and C line in Example 1 are colored, which is determined to be positive, indicating that Example 1 can detect Salmonella, and the strip is clear and dark in color, indicating that the amplification efficiency is high. However, in the colloidal gold immunochromatographic test strip in Comparative Example 1, the T line does not develop color and the C line develops color, which is determined to be negative. This shows that Comparative Example 1 cannot detect Salmonella. Although Comparative Example 2 also detects Salmonella, the strip color is light, indicating that the amplification efficiency is low.

[0071] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0072] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A microfluidic device for detecting Salmonella, characterized in that: It includes a top cover, a functional panel and a heating base which are detachably connected from top to bottom; The top cover is provided with an observation window (11), a first centrifugal hole (7), a first sample addition port (9) and a second sample addition port (10); A detection chamber (4) is provided on the functional board at a position corresponding to the observation window (11); an amplification chamber (1) is provided on the functional board and is connected to the second sample loading port (10); and a buffer chamber (2) is provided on the functional board and is connected to the first sample loading port (9); The amplification chamber (1) is reserved with a space for filling a freeze-dried reagent; the freeze-dried reagent contains a Salmonella-specific primer; The amplification chamber (1) is connected to the buffer chamber (2), the buffer chamber (2) is connected to the detection chamber (4), and a space for placing a colloidal gold immunochromatography test strip is reserved in the detection chamber (4); The functional plate is also provided with a ventilation channel (8), one end of the ventilation channel (8) being connected to the amplification chamber (1), and the other end of the ventilation channel (8) being connected to the detection chamber (4); A second centrifugal hole (6) is provided on the functional plate at a position corresponding to the first centrifugal hole (7), and the first centrifugal hole (7) and the second centrifugal hole (6) are connected; The heating base is used to be connected to a power source.

2. The microfluidic device for detecting Salmonella according to claim 1, characterized in that: A first sealing cover (3) is movably mounted on the first sample loading port (9), and a second sealing cover (5) is movably mounted on the second sample loading port (10).

3. The microfluidic device for detecting Salmonella according to claim 1, characterized in that: A heating plate is arranged on the heating base.

4. The microfluidic device for detecting Salmonella according to claim 1, characterized in that: The amplification chamber (1) and the buffer chamber (2) are connected via a first connecting channel (13), and the buffer chamber (2) and the detection chamber (4) are connected via a second connecting channel (12).

5. A method for detecting Salmonella, characterized in that: The method for detecting Salmonella is based on the microfluidic device for detecting Salmonella according to claim 1, and comprises the following steps: The test strip is prepared according to the preparation steps of the colloidal gold immunochromatographic test strip; the prepared test strip includes a detection line T line and a quality control line C line, wherein the T line is loaded with streptavidin and the C line is loaded with goat anti-mouse antibody; Preparation for detection: a freeze-dried reagent containing Salmonella-specific primers is added to the amplification chamber (1), a PBS buffer for stabilizing the pH value of the reaction system is added to the buffer chamber (2) through the first sample addition port (9), and a test strip is placed in the detection chamber (4); Adding the lysate of the sample to be tested into the second sample loading port (10), and allowing the lysate to reach the amplification chamber (1) through the second sample loading port (10), sealing the second sample loading port (10), and heating the amplification chamber (1) to 37° C. to 42° C. using the heating base, and maintaining the heating for 30 min to 40 min to perform an RPA amplification reaction, thereby obtaining an amplification product; The first centrifugal hole (7) and the second centrifugal hole (6) are fixed by a fixing belt, and then the amplification product in the amplification chamber (1) is transferred to the buffer chamber (2) for dilution by using centrifugal force. The diluted amplification product then flows into the detection chamber (4) to contact the test strip and be detected. The test strip is observed through the observation window (11) and judgment is made. The judgment standard is: if both the T line and the C line are colored positive, it means that Salmonella is detected; if the T line is not colored and the C line is colored negative, it means that Salmonella is not detected.

6. The method for detecting Salmonella according to claim 5, characterized in that: The Salmonella-specific primers include an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.

2.

7. The method for detecting Salmonella according to claim 5, characterized in that: The lyophilized reagent contains the following components at final concentrations: 520 ng / μL to 725 ng / μL recombinase, 100 ng / μL to 200 ng / μL single-stranded DNA binding protein, 350 ng / μL to 450 ng / μL DNA polymerase, 200 nM to 400 nM upstream primer, 200 nM to 400 nM downstream primer, 4% to 5% polyethylene glycol by mass fraction, 18 mM to 20 mM tris(hydroxymethyl)aminomethane hydrochloride, 180 mM to 200 mM sodium acetate, 8 mM to 10 mM dithiothreitol, 8 mM to 10 mM adenosine triphosphate, 90 mM to 100 mM creatine phosphate disodium salt, 100 ng / μL to 120 ng / μL phosphatase, 480 uM to 500 uM deoxyribonucleoside triphosphate, mass fraction 9%~10% trehalose, 40 ng / μL~50 ng / μL mannitol.

8. A primer pair for detecting Salmonella, characterized in that: It comprises the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2 in claim 5.

9. Use of a primer pair for detecting Salmonella in preparing a reagent for identifying Salmonella, characterized in that: The primer pair for detecting Salmonella is the primer pair according to claim 8, and the Salmonella is Salmonella enterica.

10. Use of the primer pair for detecting Salmonella according to claim 9 in preparing a reagent for identifying Salmonella, characterized in that: The reagent can specifically distinguish Salmonella from Salmonella, Staphylococcus aureus, Staphylococcus waldenii, Micrococcus luteus, Escherichia coli and Listeria monocytogenes.

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