Synchronous on-site detection method for multiple bacteria and application of synchronous on-site detection method

Abstract

The invention provides a synchronous on-site detection method for multiple bacteria and application of the synchronous on-site detection method, and belongs to the technical field of biological detection. Synchronous rapid detection and result judgment of various bacteria can be realized through transfer of the detector in the 8-row pipe in which the DNA extraction functional liquid and the LAMP detection liquid are preset and color change of the detection liquid pipe, so that the requirements on professional instruments are greatly reduced, the operation is simple and rapid, the labor cost and the time cost are greatly saved, and the method is suitable for a low-resource environment and has a wide application prospect. The whole process can be operated on site. The shortest time from sample collection to detection results is about 30 minutes, so that the detection speed is increased. Compared with the prior art, the method has the advantages that the operation is simple, on-site operation can be realized, the flexibility and flux of pathogenic bacteria detection are greatly improved, seven types of bacteria can be synchronously detected at the same time, and the detection efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for synchronous on-site detection of multiple bacteria and its application. Background Art

[0002] Pathogenic bacteria are an important factor affecting the healthy development of the aquaculture industry. There are many types of pathogenic bacteria in aquatic animals. Through the detection and analysis of pathogenic bacteria, the degree of risk they may cause to the aquaculture industry can be assessed, including the spread of the disease, the scope of infection, and the degree of harm to farmed animals.

[0003] Traditional pathogen identification methods mainly include plate culture technology for bacteria and microscopic examination technology for ciliates. Although these technologies are simple to operate and have intuitive results, they have problems such as long identification time and incomplete detection. With the rapid development of modern molecular biotechnology, nucleic acid-based in vitro diagnostic methods, such as 16S sequencing and PCR diagnosis, have advantages such as high modification flexibility and high detection sensitivity compared to other analytical methods based on enzymes or antibodies, and play an increasingly important role in the field of disease detection and prevention and control. In particular, isothermal polymerase amplification technology (such as LAMP, etc.) combined with microfluidic chip technology integrates all detection steps and realizes the miniaturization and portability of equipment. However, these technologies also have many common bottlenecks: for example, nucleic acid preparation is cumbersome, time-consuming, and has low throughput, relies on professional high-precision instruments, is expensive, and is limited to laboratory use. However, there is a lack of rapid and practical on-site detection methods for pathogens, resulting in untimely and inefficient analysis of pathogenic pathogens, inefficient disease prevention and control, drug abuse, and deterioration of aquaculture ecology, which brings significant economic losses to aquaculture.

[0004] Therefore, solving the cumbersome and time-consuming nucleic acid extraction problem of in vitro diagnostic technology and establishing a high-throughput, low-cost on-site pathogen diagnosis technology that does not rely on laboratory professional equipment is an inevitable trend in pathogen monitoring and a key technology that needs to be solved for rapid diagnosis and prevention of aquatic animal diseases. Based on this, it is of great significance to develop a high-throughput pathogen detection method that is simple and fast to operate from sampling to the determination of pathogenic microorganism detection results, has low equipment dependence, and is oriented to low resources and detection sites. Summary of the invention

[0005] The purpose of the present invention is to provide a method for synchronous on-site detection of multiple bacteria and its application, which is simple and fast to operate, greatly saves labor costs and time costs, is suitable for low-resource environments, and the entire process can be operated on-site. From the collection of samples to the detection results, it only takes about 30 minutes at the shortest, which improves the speed of detection.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for synchronous on-site detection of multiple bacteria, comprising the following steps:

[0008] A method for synchronous on-site detection of multiple bacteria comprises the following steps:

[0009] (1) preparing functional tanks I, II, III and IV with functional liquids I, II, III and IV and detectors respectively;

[0010] (2) After the sample to be tested is added to the functional tank I and allowed to stand, the detector is inserted into the tank to contact with the functional liquid I;

[0011] (3) inserting the detector after step (2) into the functional tank II, immersing the functional area of ​​the detector in the functional liquid II, and then taking it out and drying it;

[0012] (4) transferring the detector after step (3) to functional tank III or IV, so that the functional area of ​​the detector is in contact with functional liquid III or IV;

[0013] (5) Add the functional liquid III obtained in step (4) into the functional tank IV and mix;

[0014] (6) The functional tank IV having completed step (4) or (5) is placed at 58 to 68° C. for 15 to 60 minutes, and the test result is determined based on the color change of the functional liquid IV.

[0015] Preferably, the functional liquid I comprises one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Chelex, proteinase K, NaOH, NaHCO3, NaCl, EDTA, Tris-HCl, mercaptoethanol, and dithiothreitol;

[0016] The functional liquid II comprises one or both of an ethanol aqueous solution and an isopropanol aqueous solution, and the volume fraction of the functional liquid II is 65%-80%;

[0017] The functional liquid III is one of water, PBS buffer and TE buffer;

[0018] The functional liquid IV includes BstDNA polymerase, LAMP primer set and indicator.

[0019] Preferably, the concentration of sodium dodecyl sulfate in the functional solution I is 0.5-20%, the concentration of hexadecyltrimethylammonium bromide is 0.01-0.05M, the concentration of Che1ex is 0.5-20%, the concentration of proteinase K is 5-30μg / mL, the concentration of NaCl is 0.05-2M, the concentration of EDTA is 0.01-0.05M, the concentration of Tris-HCl is 20-500mM, the concentration of mercaptoethanol is 0.05-0.5%, and the concentration of dithiothreitol is 0.2-5M;

[0020] The LAMP primer group in the functional liquid IV includes two or three groups of EIP and BIP group, F3 and B3 group, LP and LB group; the concentration of EIP / BIP is 8-16 μM, the concentration of F3 / B3 is 0.5-2 μM, and the concentration of LP / LB is 2-8 μM;

[0021] The concentration of the BstDNA polymerase is 0.2-1M, and the indicator is calcein and hydroxy One or a mixture of two phenol blues, the concentration of calcein is 5 to 50 μM; the concentration of hydroxynaphthol blue is 50 to 500 μM.

[0022] Preferably, in step (1), the detector is a functional body comprising a polymer functional area with nucleic acid adsorption function and a handle area for transfer; the material of the functional area comprises one or more of polyacrylic acid, polyethylene glycol diacrylate, polydopamine, and silica.

[0023] Preferably, in step (2), the standing time is 10 to 30 minutes, the temperature is 5 to 70° C.; the contact time is 10 seconds to 5 minutes, and the sample includes animal tissue, cells, bacteria, and filtered water membrane.

[0024] Preferably, in step (2), the detector is inserted into the slot to contact with the functional liquid I, and one or two of the auxiliary solutions of anhydrous ethanol and isopropanol are added during the contact.

[0025] Preferably, in step (3), the contact time is 10 to 60 seconds per time, and the drying time is 1 to 10 minutes.

[0026] Preferably, in step (4), the contact time is 5 s to 5 min.

[0027] The present invention also provides an application of a multi-bacteria synchronous on-site detection method in detecting pathogens.

[0028] The beneficial effects of the present invention compared with the prior art are:

[0029] (1) The present invention can realize the simultaneous rapid detection and result determination of multiple bacteria through the transfer of the detector in the 8-tube strip pre-filled with DNA extraction functional solution and LAMP detection solution and the color change of the detection solution tube, which greatly reduces the demand for professional instruments, is simple and fast to operate, greatly saves labor costs and time costs, is suitable for low-resource environments, and the whole process can be operated on-site. From the collected sample to the test result, it only takes about 30 minutes at the shortest, which improves the speed of detection.

[0030] (2) Compared with the prior art, the present invention is simple to operate and can be applied on site. The flexibility and throughput of detecting pathogenic bacteria are greatly improved. Up to 7 types of bacteria can be detected simultaneously, and the detection efficiency is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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 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 labor.

[0032] Figure 1 The detector prepared in Example 1, wherein a is the detector obtained by the first preparation method, and wherein b is the detector obtained by the second preparation method;

[0033] Figure 2 The results of the 7-linked bacterial synchronous detection in Example 1;

[0034] Figure 3 These are the detection results of Vibrio splendens at gradient concentrations in Example 2, wherein a is the detection result under natural light, and b is the detection result under ultraviolet light. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Table 1 LAMP primer sequence list used in the present invention

[0041]

[0042]

[0043]

[0044] The present invention provides a method for synchronous on-site detection of multiple bacteria, comprising the following steps:

[0045] (1) preparing functional tanks I, II, III and IV with functional liquids I, II, III and IV and detectors respectively;

[0046] (2) After the sample to be tested is added to the functional tank I and allowed to stand, the detector is inserted into the tank to contact with the functional liquid I;

[0047] (3) Insert the detector that has completed step (2) into the functional tank II, immerse the functional area of ​​the detector in the functional liquid II, shake it up and down, then take it out and dry it;

[0048] (4) transferring the detector after step (3) to functional tank III or IV, so that the functional area of ​​the detector is in contact with functional liquid III or IV;

[0049] (5) Add the functional liquid III obtained in step (4) into the functional tank IV and mix;

[0050] (6) The functional tank IV having completed step (4) or (5) is placed at 58 to 68° C. for 15 to 60 minutes, and the test result is determined based on the color change of the functional liquid IV.

[0051] Preferably, the functional liquid I comprises one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Chelex, proteinase K, NaOH, NaHCO3, NaCl, EDTA, Tris-HCl, mercaptoethanol, and dithiothreitol;

[0052] The functional liquid II comprises one or both of an ethanol aqueous solution and an isopropanol aqueous solution, and the volume fraction of the functional liquid II is 65%-80%;

[0053] The functional liquid III is one of water, PBS buffer and TE buffer;

[0054] The functional liquid IV includes BstDNA polymerase, LAMP primer set and indicator.

[0055] Preferably, the concentration of sodium dodecyl sulfate in the functional solution I is 0.5-20%, the concentration of hexadecyltrimethylammonium bromide is 0.01-0.05M, the concentration of Chelex is 0.5-20%, the concentration of proteinase K is 5-30μg / mL, the concentration of NaCl is 0.05-2M, the concentration of EDTA is 0.01-0.05M, the concentration of Tris-HCl is 20-500mM, the concentration of mercaptoethanol is 0.05-0.5%, and the concentration of dithiothreitol is 0.2-5M;

[0056] The LAMP primer group in the functional fluid IV includes two or three groups of EIP and BIP group, F3 and B3 group, LP and LB group; the concentration of EIP / BIP is 8-16μM, the concentration of F3 / B3 is 0.5-2μM, and the concentration of LP / LB is 2-8μμM.

[0057] The concentration of the BstDNA polymerase is 0.2-1M, and the indicator is calcein and hydroxy One or a mixture of two phenol blues, the concentration of calcein is: 5-50 μM; hydroxy The concentration of phenol blue is 50-500 μM.

[0058] Preferably, in step (1), the detector is a functional body comprising a polymer functional area with nucleic acid adsorption function and a handle area for transfer; the material of the functional area comprises one or more of polyacrylic acid, polyethylene glycol diacrylate, polydopamine, and silica; the shapes of the various parts of the detector are not restricted and can be flexibly adjusted according to the usage scenario.

[0059] Preferably, in step (2), the standing time is 10 to 30 minutes, the temperature is 5 to 70°C; the shaking time of the detector is 10 seconds to 5 minutes, and the sample includes animal tissue, cells, bacteria, filtered water filter membrane

[0060] Preferably, in step (2), the detector is inserted into the tank to contact with the functional liquid I, and one or two of the auxiliary solutions of anhydrous ethanol and isopropanol are added during the contact.

[0061] Preferably, the drying in step (3) is not limited in form, and can be dried naturally or heated, and the time is also not limited, preferably 1 to 10 minutes at room temperature.

[0062] Preferably, the functional tank II comprises one or more groups, preferably 1 to 3 groups, which can allow the detector to contact with the functional liquid II 1 to 3 times, with the contact time being 10 to 60 seconds per time.

[0063] Preferably, in step (4), the contact time is 5 s-5 min.

[0064] Preferably, the functional liquid III obtained in step (4) is added to the functional tank IV in a volume of 0.2 to 5 μL.

[0065] Example 1

[0066] Embodiment 1 of the present invention provides a method for simultaneous on-site detection of multiple bacteria, and the specific steps are as follows:

[0067] (1) Preparation of detector:

[0068] Method 1: The structure of the joint detector was designed using Solidworks modeling software, and 5wt% of nano-SiO2 was doped with commercial acrylic resin. The separator was prepared by DLP 3D printing with a printing layer thickness of 50μm. The functional area size was a cone with a threaded structure of 2.0mm radius and 10.00mm height. The handle was an integrally printed slender cylinder with a height of 50mm. The prepared detector was repeatedly cleaned with deionized water and anhydrous ethanol, and dried for use. ( Figure 1 a)

[0069] The second method: Use a commercially available 0.2mL or 1.5mL centrifuge tube as a mold, pour about 1cm of prepolymer solution (monomer is acrylic acid, containing 50wt% quartz sand particles (40-270μm), 0.02wt% cross-linking agent, 24.995wt% initiator) into the mold, insert a toothpick, put the mold and prepolymer solution under ultraviolet light, use light to initiate free radical polymerization, the ultraviolet light wavelength is 280-400nm, irradiate for 3-10 minutes on all sides to completely cure it, clean the detector with deionized water and anhydrous ethanol, and dry it for later use. ( Figure 1 b)

[0070] (2) Preparation of functional groove:

[0071] Add 100 μL of functional solution I (0.1 M Tris-HCl, 25 mM of EDTA, 50 mM of NaCl, 1 wt% SDS, 10 μg / mL proteinase K) to each of the eight strip tubes to make functional tank I;

[0072] Add 150 μL of functional solution II (75% anhydrous ethanol) to each of the two sets of 8-tube strips placed side by side to make functional tank II;

[0073] Add 80 μL of functional solution III (TE buffer) to each well of the eight-tube strip to make functional reservoir III;

[0074] Add 23 μL of functional solution IV (2×BcaBEST Buffer 12.5 μL, EIP 0.4 μL, BIP 0.4 μL, F3 0.1 μL, B3 0.1 μL, LP 0.2 μL, LB 0.2 μL, indicator 0.85 μL) to each well of the eight-tube strip to make functional well IV;

[0075] (3) Multiple bacterial detection

[0076] In the 1#-7# tubes of the functional tank I, add about 1 mg of Vibrio splendidus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio rotiferus, Vibrio corallolyticus, and Shewanella, and place for 10 minutes until the sample tissue is completely dissolved and the solution becomes clear; add 70 μL of anhydrous ethanol to the lysis tank centrifuge tubes of the 1#-8# wells, insert the detector prepared by the first method, and immerse the functional area of ​​the detector in the liquid of the 1-8 wells respectively, and shake the detector for 0.5 mm; transfer the detector to the first group of slots of the functional tank, immerse the functional area of ​​the detector under the liquid level of each well, shake the detector up and down for 30 seconds, then take out the detector, transfer the detector to the second group of functional tanks II, and repeat the above operation; remove the detector, air dry it at room temperature for 5 minutes, and then transfer it to the functional tank III, shake it up and down for 1 minute, and remove the detector. Take 2 μL each and add it to each well of the functional tank IV.

[0077] Among them, primer groups 1-7# target Vibrio brilli, Vibrio rotifera, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio corallilyticus and Vibrio shewani, respectively. Cover the eight-tube strip and place it at 62°C for 25 minutes. Put the heating pack into the water, heat the water to boiling, transfer the eight-tube strip to the heated water for 2 minutes, and judge the result according to the color of functional fluid IV ( Figure 2 ).

[0078] Figure 2 The display shows that the eight-row tubes 1-7# are the detection holes for Vibrio splendidus, Vibrio rotiferus, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio corallolyticus, and Vibrio Shewanella, and the 8# hole is a negative reference. Under natural light, holes 1-7 appear blue and hole 8 appears purple, indicating that the detection results of holes 1-7 are positive for Vibrio splendidus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio rotiferus, Vibrio corallolyticus, and Shewanella.

[0079] Test Example 1

[0080] Test Example 1 of the present invention detects the sensitivity of the multi-bacteria synchronous on-site detection method, and the specific steps are as follows:

[0081] In a solution containing 200 μl of functional solution I (0.1 M Tris-HCl, 25 mM EDTA, 50 mM NaCl, 1 wt% SDS, 10 μg / mL proteinase K, 5 μg / mL RNA digestion enzyme) in 1.5mL centrifuge tubes 1-4#, gradient Vibrio splendidus cells (bacterial concentrations are original concentration, 103CFU / mL, 100CFU / mL and 100CFU / mL, respectively), placed at 60°C for 20min, 150μL of anhydrous ethanol is added to each tube in the lysis tank centrifuge tube, and the detector prepared by the second method is inserted, the functional area of ​​the detector is immersed in the liquid, and the detector is gently shaken for 1.5min; the detector is transferred to the first group 1-4# tubes of functional tank II pre-filled with 300μL functional liquid II (75% isopropanol), the functional area of ​​the detector is immersed under the liquid surface, the detector is gently shaken up and down for 30s, then the detector is taken out and placed in the second and third groups 1-4# tubes of functional tank II in turn to repeat the above operation; the detector is removed, air-dried at room temperature for 10min, and then transferred to the functional tank III containing 80μL of functional liquid III (75% ethanol), and shaken up and down for 2min. Take 2 μL of each and add them to new 0.2 mL centrifuge tubes 1-4# pre-filled with 23 μL functional solution IV (2×BcaBEST Buffer 12.5 μL, Vsp-EIP 0.4 μL, Vsp-BIP 0.4 μL, Vsp-F3 0.1 μL, Vsp-B3 0.1 μL, Vsp-LP 0.2 μL, Vsp-LB 0.2 μL, indicator 0.85 μL, water 0.25 μL). In centrifuge tube 5#, replace the added functional solution III with 2 μL of pure water. After placing the functional tank IV in a 62° constant temperature module for 25 min, transfer the functional tank IV to a 90°C heating module and let it stand for 2 min. Determine the result based on the color of the functional solution IV ( Figure 3 ).

[0082] Explanation of the results: 1-4# are the test wells for Vibrio splendens at the original concentration, 103 CFU / mL, 100 CFU / mL and 100 CFU / mL respectively, and well 5# is the negative reference. Figure 3 a. Under natural light, 1-4# appear blue, 5# appears purple, such as Figure 3 b. Under ultraviolet light, 1-3# appears green, 4# appears light pink, and 5# appears pink, indicating that the test results of 1-3# are positive for Vibrio splendens, and 4# is weakly positive for Vibrio splendens.

[0083] It can be seen that the detection limit of the technology of the present invention is <100 CFU / mI, and the detection sensitivity is high.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneous on-site detection of multiple bacteria, characterized in that: The steps include: (1) preparing functional tanks I, II, III and IV with functional liquids I, II, III and IV and detectors respectively; (2) After the sample to be tested is added to the functional tank I and allowed to stand, the detector is inserted into the tank to contact with the functional liquid I; (3) Insert the detector that has completed step (2) into functional slot II, immerse the functional area of ​​the detector in functional liquid II, then take it out and dry it; (4) transferring the detector after step (3) to functional tank III or IV, so that the functional area of ​​the detector is in contact with functional liquid III or IV; (5) Add the functional liquid III obtained in step (4) into the functional tank IV and mix; (6) Place the functional tank IV that has completed step (4) or (5) at 58-68°C for 15-60 min, and determine the test result based on the color change of the functional liquid IV.

2. The method for simultaneous on-site detection of multiple bacteria according to claim 1, characterized in that: The functional liquid I comprises one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Chelex, proteinase K, NaOH, NaHCO3, NaCl, EDTA, Tris-HCl, mercaptoethanol, and dithiothreitol; The functional liquid II includes one or both of an ethanol aqueous solution and an isopropanol aqueous solution, and the volume fraction of the functional liquid II is 65%-80%; The functional liquid III is one of water, PBS buffer and TE buffer; The functional liquid IV includes Bst DNA polymerase, LAMP primer set and indicator.

3. The method for simultaneous on-site detection of multiple bacteria according to claim 2, characterized in that: In the functional solution I, the concentration of sodium dodecyl sulfate is 0.5-20%, the concentration of hexadecyltrimethylammonium bromide is 0.01-0.05 M, the concentration of Chelex is 0.5-20%, the concentration of proteinase K is 5-30 µg / mL, the concentration of NaCl is 0.05-2M, the concentration of EDTA is 0.01-0.05 M, the concentration of Tris-HCl is 20-500 mM, the concentration of mercaptoethanol is 0.05-0.5%, and the concentration of dithiothreitol is 0.2-5 M; The LAMP primer group in the functional liquid IV includes two or three groups of EIP and BIP group, F3 and B3 group, LP and LB group; the concentration of EIP / BIP is 8-16 µM, the concentration of F3 / B3 is 0.5-2 µM, and the concentration of LP / LB is 2-8 µM; The concentration of the Bst DNA polymerase is 0.2-1M, the indicator is one or a mixture of calcein and hydroxynaphthol blue, the concentration of calcein is 5-50 μM, and the concentration of hydroxynaphthol blue is 50-500 μM.

4. The method for simultaneous on-site detection of multiple bacteria according to claim 1, characterized in that: In step (1), the detector is a functional body comprising a polymer functional area with nucleic acid adsorption function and a handle area for transfer; the material of the functional area comprises one or more of polyacrylic acid, polyethylene glycol diacrylate, polydopamine, and silicon dioxide.

5. The method for simultaneous on-site detection of multiple bacteria according to claim 1, characterized in that: In step (2), the standing time is 10-30 min, the temperature is 5-70°C; the contact time is 10 s-5 min, and the sample includes animal tissue, cells, bacteria, and filtered water membrane.

6. The method for simultaneous on-site detection of multiple bacteria according to claim 1, characterized in that: In step (2), the detector is inserted into the tank to contact with the functional liquid I, and one or two of the auxiliary solutions of anhydrous ethanol and isopropanol are added during the contact.

7. The method for simultaneous on-site detection of multiple bacteria according to claim 1, characterized in that: In step (3), the contact time is 10 to 60 seconds per time, and the drying time is 1 to 10 minutes.

8. The method for simultaneous on-site detection of multiple bacteria according to claim 1, characterized in that: The contact time in step (4) is 5 s to 5 min.

9. Application of the multi-bacteria synchronous on-site detection method according to any one of claims 1 to 8 in pathogen detection.

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