Method for rapidly detecting coliform

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a method for rapidly detecting coliforms, which comprises the following steps: (1) mixing a sample to be detected with a diluent to obtain a sample solution; (2) centrifuging the sample liquid to remove supernate, and uniformly mixing precipitate with a culture medium to obtain suspension liquid; the culture medium is an EC-MUG culture medium or a mixture of an EC culture medium and FDG; (3) generating water-in-oil liquid drops from the suspension through a microfluidic liquid drop technology; (4) culturing the liquid drops in an incubator for a period of time; and (5) scanning the cultured liquid drops by using exciting light, detecting the total number of the liquid drops and the fluorescence intensity of each liquid drop, and then calculating the concentration of the coliform bacteria by using a Poisson distribution formula. According to the invention, the coliform group is divided into single bacterium liquid drops by adopting a microdroplet generation technology, and the total number of the coliform group is detected by utilizing an enzyme substrate luminescence principle and a fluorescence scanning counting technology, so that the rapid and accurate detection of the coliform group is realized.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a method for rapid detection of coliform bacteria. Background Technology

[0002] Coliform bacteria are aerobic and facultative anaerobic Gram-negative non-spore-forming bacilli that can ferment lactose, produce acid and gas under certain culture conditions. They are commonly used as microbial contamination indicators for water quality and food hygiene. The quantity of coliform bacteria indicates the degree of fecal contamination in the environment or the risk of pathogens.

[0003] Currently, the number of coliform bacteria is usually detected using the MPN method (most probable number method) and plate count method specified in the national standard GB 4789.3-2025 (National Food Safety Standard for Microbiological Examination of Food - Coliform Count). The flowchart is shown below. Figure 1 and Figure 2 As shown. However, the following drawbacks exist:

[0004] 1) Long detection time: MPN method or plate counting method requires 48h-96h;

[0005] 2) The plate count method has inherent errors: The standard specifies that 1 mL of sample homogenate is pipetted into a sterile petri dish, then 15 mL–20 mL of VRBA medium cooled to approximately 48°C is poured into the dish. The medium is mixed with the sample, allowed to solidify at room temperature, and then incubated. The colonies growing on the surface are then counted. This method can cause multiple bacteria to aggregate, resulting in overlapping colonies and thus multiple bacteria being counted as a single colony. Furthermore, this method may result in some microorganisms growing within the agar medium, which are not visible to the naked eye, leading to errors in the actual number of microorganisms in the sample.

[0006] 3) Errors exist in colony counting methods: The national standard method stipulates that "the naked eye observation method is used, sometimes with a magnifying glass or colony counter, and the dilution factor and the corresponding number of colonies are recorded." The errors caused by this manual counting method can also lead to inaccurate experimental results.

[0007] 4) Errors exist in the MPN method: MPN is a quantitative detection method that combines statistics and microbiology. After the sample is serially diluted and cultured, the most probable number of coliform bacteria in the sample is calculated using statistical probability theory based on the lowest dilution where no growth occurs and the highest dilution where growth occurs. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a method for rapid detection of coliform bacteria, comprising the following steps:

[0009] (1) Mix the sample to be tested with the diluent to obtain the sample solution;

[0010] (2) Centrifuge the sample solution to remove the supernatant, and mix the precipitate with the culture medium to obtain a suspension; the culture medium is EC-MUG medium, or a mixture of EC medium and FDG;

[0011] (3) The suspension is used to generate water-in-oil droplets using microfluidic droplet technology, and the droplet size is 25-100 μm;

[0012] (4) Place the droplet in an incubator and incubate for a period of time to allow the coliform bacteria in the droplet to proliferate rapidly and selectively decompose MUG or FDG to release fluorescent substances.

[0013] (5) Scan the cultured droplets with excitation light having the maximum excitation wavelength, and detect the total number of droplets and the fluorescence intensity of each droplet at the maximum emission wavelength. Then calculate the coliform concentration using the Poisson distribution formula. The maximum excitation wavelength and the maximum emission wavelength are determined according to the characteristics of the released fluorescent substance. The maximum excitation wavelength is consistent with the peak absorption wavelength of the fluorescent substance.

[0014] This invention involves centrifuging a sample solution containing the test sample to remove the supernatant. The resulting precipitate is mixed with a culture medium, and then the culture medium system containing coliform bacteria is divided into microdroplets using microfluidic technology. Each droplet contains a very low concentration of coliform bacteria and either MUG (4-methylumbelliferone-β-D-glucuronide) or FDG (fluorescein 2-β-D-galactopyranoside). Through a short period of incubation in the microdroplets, the coliform bacteria proliferate and selectively decompose MUG or FDG, releasing fluorescent substances. 4-Methylumbelliferone (or fluorescein) is used to detect fluorescence through specific excitation and emission spectra. The fluorescence threshold is set at the average fluorescence intensity + 10 times the standard deviation. Droplets with fluorescence intensity above the threshold are considered positive droplets, and those below the threshold are considered negative droplets. Based on the proportion of positive droplets in the total number of droplets (λ = -ln(1-p), where λ is the average number of bacteria per droplet and p is the proportion of positive droplets), the total number of coliform bacteria in the original droplets is calculated using the Poisson distribution formula. This method achieves absolute quantitative detection of coliform bacteria.

[0015] This invention utilizes microfluidic droplet technology to generate water-in-oil droplets with a diameter of 25–100 μm. When the droplet size is too large, the large droplets easily break into multiple smaller droplets, affecting droplet uniformity and leading to decreased detection precision. Furthermore, large droplets have a slow flow rate during detection, resulting in slower detection speed. When the droplet size is too small, on the one hand, the fluorescence signal weakens, requiring excessively high sensitivity from the fluorescence detector; on the other hand, for the same sample volume, the total number of droplets increases, while the detector has an upper limit on the number of droplets it can identify per second, leading to excessively long detection times. Reading times for droplets smaller than 25 μm exceed 3 hours, making them unsuitable for rapid detection; for droplets larger than 100 μm, the detection limit is higher than 10 cfu / ml.

[0016] Preferably, in step (1), the diluent is sterile phosphate buffer or sterile saline.

[0017] When the sample to be tested is a liquid sample, the volume ratio of the sample to the diluent is 1:3 to 10;

[0018] When the test sample is a solid sample, the mass-to-volume ratio of the test sample to the diluent is 1g:3-10mL.

[0019] Preferably, in step (2), the centrifugation speed is 5000-10000×g and the time is 5-10min.

[0020] Preferably, in step (2), the volume of the culture medium is 75 μL to 225 μL, with the sample solution volume being 1 mL. Studies have found that if the proportion of liquid culture medium is too low, it is greatly affected by the sample matrix, leading to slower bacterial growth or even death, thus affecting the detection results. If the proportion of culture medium is too high, the sample solution is diluted excessively, resulting in a higher detection limit, lower detection sensitivity, and loss of practical value.

[0021] Preferably, in step (2), when the culture medium is EC-MUG medium, the mass fraction of MUG in the EC-MUG medium is 0.002 to 0.010%;

[0022] When the culture medium is a mixture of EC medium and FDG, the mass ratio of EC medium to FDG is 1:0.00002–0.0001. Studies have found that a low proportion of MUG or FDG in the culture medium leads to a weaker fluorescence signal and false negatives. An excessively high proportion leads to stronger background fluorescence, which easily produces false positives and false negatives, and reduces detection sensitivity.

[0023] As a preferred embodiment, in step (3), the specific process of the microfluidic droplet technology is as follows: using the droplet-generating oil as the oil phase and the suspension as the water phase, the water phase and the oil phase are injected into the channels 1 and 2 of the microfluidic droplet generator respectively, and the pressure in the channels 1 and 2 is controlled by the microfluidic droplet generation to obtain water-in-oil droplets.

[0024] Preferably, in step (4), the culture time is 3 to 5 hours.

[0025] Preferably, when the culture medium is EC-MUG medium, the maximum excitation wavelength is 355-375 nm and the maximum emission wavelength is 440-460 nm;

[0026] When the culture medium is a mixture of EC medium and FDG, the maximum excitation wavelength is 490-492 nm and the maximum emission wavelength is 514-520 nm.

[0027] The beneficial effects of this invention are as follows:

[0028] 1) The present invention provides a method for rapid detection of coliform bacteria, which uses microdroplet culture to culture coliform bacteria. The individual coliform bacteria in the microdroplet form a self-contained system and are not disturbed by other bacteria. At the same time, the small droplet culture system can accelerate the delivery of nutrients and make it easier for coliform bacteria to utilize nutrients, so that coliform bacteria can rapidly proliferate and grow in a short period of time and the culture time is greatly shortened.

[0029] 2) The present invention provides a method for rapid detection of coliform bacteria, in which coliform bacteria are independently distributed in individual droplets, making each coliform bacteria a separate system, and the individual coliform bacteria in the sample are more dispersed, avoiding the phenomenon of colony overlap, and the counting is accurate;

[0030] 3) The present invention provides a method for rapid detection of coliform bacteria, which improves the accuracy and sensitivity of detection results by optimizing the amount of culture medium and the ratio of MUG / FDG therein;

[0031] 4) The present invention provides a method for rapid detection of coliform bacteria, which obtains fluorescence signals by instrument scanning and calculates the number of coliform bacteria by absolute quantification using the Poisson distribution method, resulting in more accurate results and significantly shortening the counting and detection time. Attached Figure Description

[0032] Figure 1 The test procedure is based on the MPN counting method for coliform bacteria, as specified in GB 4789.3-2025.

[0033] Figure 2 The procedure is based on the plate count method for coliform bacteria as specified in GB 4789.3-2025.

[0034] Figure 3 This is a droplet fluorescence intensity diagram from Example 1.

[0035] Figure 4 This is a peak width and peak height diagram of the total number of droplets in Example 1.

[0036] Figure 5 This is a diagram showing the droplet peak area and peak height of Example 1.

[0037] Figure 6 This is a droplet fluorescence intensity diagram from Example 2.

[0038] Figure 7 This is a peak width and peak height diagram of the total number of droplets in Example 2.

[0039] Figure 8 This is a diagram showing the droplet peak area and peak height in Example 2. Detailed Implementation

[0040] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0041] In the following examples, the formulation of the phosphate buffer used is as follows: 8.0 g sodium chloride (NaCl), 0.20 g potassium chloride (KCl), 0.24 g potassium dihydrogen phosphate (KH2PO4), 1.44 g disodium hydrogen phosphate (Na2HPO4), and 1000 mL distilled water.

[0042] In the following examples, the EC-MUG medium used was obtained from Beijing Luqiao Technology Co., Ltd., and the mass fraction of MUG in the EC-MUG medium was 0.005%.

[0043] In the following examples, the EC culture medium used was obtained from Beijing Luqiao Technology Co., Ltd.

[0044] In the following examples, the FDG used was sourced from MedChemexpress Biotechnology, Inc., USA.

[0045] In the following examples, the microfluidic droplet generator used is a microfluidic droplet preparation instrument, model DC400, from Zhejiang Dapu Biotechnology Co., Ltd.

[0046] In the following examples, the scanning detector used is a high-throughput droplet sorter, model DS1200, from Zhejiang Dapu Biotechnology Co., Ltd.

[0047] Example 1

[0048] A rapid method for detecting coliform bacteria, comprising the following steps:

[0049] 1. Sample preparation: Escherichia coli (CMCC 44102) was inoculated into soy sauce (tested sterile) at a concentration of approximately 10. 4 The soy sauce sample was obtained by measuring CFU / mL.

[0050] 2. Sample testing: Weigh 25 mL of the soy sauce sample to be tested, place it in a sterile homogenizing cup containing 225 mL of sterile phosphate buffer, mix thoroughly, and prepare the sample solution.

[0051] 3. Generation of bacterial droplets: Take 1 mL of sample solution, centrifuge at 10000×g for 7 min, remove the supernatant, and prepare a suspension of the precipitate with 75 μL EC-MUG medium. Mix well.

[0052] The suspension was prepared into water-in-oil droplets with a particle size of 25-100 μm using a microfluidic droplet generator. Specifically, using Dapbio's universal droplet generating oil as the oil phase and the suspension as the aqueous phase, the aqueous phase and the oil phase were injected into channels 1 and 2 of the microfluidic droplet generator, respectively. The pressure in channels 1 and 2 was controlled by the microfluidic droplet generator to obtain water-in-oil droplets.

[0053] 4. Bacterial culture and staining: The prepared droplets were placed in an incubator and incubated at 36℃±1℃ for 3 hours; the microorganisms in the droplets proliferated rapidly, and Escherichia coli selectively decomposed MUG to release fluorescent substances.

[0054] 5. Scanning Counting: After cultivation, the microdroplets were fed into the instrument. Excitation / emission wavelengths of 365nm / 450nm were selected, and fluorescence values ​​were detected using a scanning detector. The total number of droplets and fluorescence intensity were measured, and the results are as follows: Figure 3-5 As shown, Figure 3 This is a droplet fluorescence intensity diagram from Example 1. Figure 3 Each fluorescence peak represents a droplet, and the calculated average fluorescence intensity of the droplets is 175.0, with a standard deviation of 9.6. The fluorescence threshold, 271.0, is set at the average fluorescence intensity plus 10 times the standard deviation. Peaks with fluorescence intensities above this threshold are considered positive droplets, while those below are considered negative droplets. Figure 5 The total number of droplets is 405,967, and the number of positive droplets is 549.

[0055] First, the average number of bacteria λ per droplet is calculated based on the proportion p of positive droplets in the total number of droplets. The result is 0.00135 (λ = -ln(1-p), where λ is the average number of bacteria per droplet and p is the proportion of positive droplets). Then, the concentration of E. coli in the sample (i.e., the total number of E. coli) is calculated using the Poisson distribution formula.

[0056] Total number of E. coli = λ × n / V3 × (V1 + V2) / V1

[0057] Where: n: total number of droplets;

[0058] V1: Volume of the soy sauce sample to be tested (25 mL);

[0059] V2: Diluent volume (225 mL);

[0060] V3: Centrifuged sample volume (1 mL);

[0061] 6. Results: From sample processing to result output, the detection was completed within 4-6 hours, and the calculated total E. coli count was 5.5 × 10⁻⁶. 3 CFU / mL, the total bacterial count of the sample was verified to be 6×10⁶ by plate culture. 3 The CFU / mL value is consistent with the order of magnitude of the above detection results.

[0062] Example 2

[0063] 1. Sample preparation: Escherichia coli (CMCC 44102) was inoculated into soy sauce (tested sterile) at a concentration of approximately 10. 4 The soy sauce sample was obtained by measuring CFU / mL.

[0064] 2. Sample testing: Weigh 25 mL of the soy sauce sample to be tested, place it in a sterile homogenizing cup containing 225 mL of sterile phosphate buffer, mix thoroughly, and prepare the sample solution.

[0065] 3. Generation of bacterial droplets: Take 1 mL of sample solution, centrifuge at 10000×g for 7 min, remove the supernatant, and prepare a suspension from the precipitate using a mixture of 75 μL EC medium and 0.3 μL FDG aqueous solution (FDG mass fraction in the aqueous solution is 1%).

[0066] The suspension was prepared into water-in-oil droplets with a particle size of 25-100 μm using a microfluidic droplet generator. Specifically, using Dapbio's universal droplet generating oil as the oil phase and the suspension as the aqueous phase, the aqueous phase and the oil phase were injected into channels 1 and 2 of the microfluidic droplet generator, respectively. The pressure in channels 1 and 2 was controlled by the microfluidic droplet generator to obtain water-in-oil droplets.

[0067] 4. Bacterial culture and staining: The prepared droplets were placed in an incubator and incubated at 36℃±1℃ for 3 hours; the microorganisms in the droplets proliferated rapidly, and Escherichia coli selectively decomposed FDG to release fluorescent substances.

[0068] 5. Scanning Counting: After cultivation, the microdroplets were fed into the instrument. Excitation / emission wavelengths of 492nm / 520nm were selected, and fluorescence values ​​were detected using a scanning detector. The total number of droplets and fluorescence intensity were determined, and the results are as follows: Figure 6-8 As shown, Figure 6 This is a droplet fluorescence intensity diagram from Example 2. Figure 6 Each fluorescence peak represents a droplet, and the average fluorescence intensity of the droplet can be calculated to be 443 with a standard deviation of 33. The fluorescence threshold, 773, is set at the average fluorescence intensity + 10 times the standard deviation. Peaks with fluorescence intensities above the threshold are considered positive droplets, and those below are considered negative droplets. Figure 8 The total number of droplets is 396,499, and the number of positive droplets is 1,059.

[0069] First, the average number of bacteria λ per droplet is calculated based on the proportion p of positive droplets in the total number of droplets. The result is 0.00267 (λ = -ln(1-p), where λ is the average number of bacteria per droplet and p is the proportion of positive droplets). Then, the concentration of E. coli in the sample (i.e., the total number of E. coli) is calculated using the Poisson distribution formula.

[0070] Total number of E. coli = λ × n / V3 × (V1 + V2) / V1

[0071] Where: n: total number of droplets;

[0072] V1: Volume of the soy sauce sample to be tested (25 mL);

[0073] V2: Diluent volume (225 mL);

[0074] V3: Centrifuged sample volume (1 mL);

[0075] 6. Results: From sample processing to result output, the detection was completed within 4-6 hours, and the calculated total coliform count was 1.1 × 10⁻⁶. 4 The bacterial concentration, measured using the traditional plate method, was 1.4 × 10⁻⁶ CFU / mL. 4 The CFU / mL value is consistent with the order of magnitude of the above detection results.

[0076] The above embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for rapid detection of coliform bacteria, characterized in that, Includes the following steps: (1) Mix the sample to be tested with the diluent to obtain the sample solution; (2) Centrifuge the sample solution to remove the supernatant, and mix the precipitate with the culture medium to obtain a suspension; the culture medium is EC-MUG medium, or a mixture of EC medium and FDG; (3) The suspension is used to generate water-in-oil droplets using microfluidic droplet technology, and the droplet size is 25-100 μm; (4) Place the droplet in an incubator and incubate for a period of time; (5) Scan the cultured droplets with excitation light at the maximum excitation wavelength, and detect the total number of droplets and the fluorescence intensity of each droplet at the maximum emission wavelength. Then calculate the coliform concentration using the Poisson distribution formula.

2. The method for rapid detection of coliform bacteria according to claim 1, characterized in that, In step (1), the diluent is sterile phosphate buffer or sterile physiological saline; When the sample to be tested is a liquid sample, the volume ratio of the sample to the diluent is 1:3 to 10; When the sample to be tested is a solid sample, the mass-to-volume ratio of the sample to the diluent is 1g:3mL to 10mL.

3. A method for rapid detection of coliform bacteria according to claim 1 or 2, characterized in that, In step (2), the centrifugation speed is 5000-10000×g and the time is 5-10min.

4. A method for rapid detection of coliform bacteria according to claim 1 or 2, characterized in that, In step (2), the volume of the sample solution is 1 mL, and the amount of the culture medium used is 75 μL to 225 μL.

5. A method for rapid detection of coliform bacteria according to claim 1 or 2, characterized in that, In step (2), when the culture medium is EC-MUG medium, the mass fraction of MUG in EC-MUG medium is 0.002% to 0.010%; When the culture medium is a mixture of EC medium and FDG, the mass ratio of EC medium to FDG is 1:0.00002 to 0.0001.

6. A method for rapid detection of coliform bacteria according to claim 1 or 2, characterized in that, In step (3), the specific process of the microfluidic droplet technology is as follows: using the droplet-generating oil as the oil phase and the suspension as the water phase, the water phase and the oil phase are injected into the channels 1 and 2 of the microfluidic droplet generator respectively, and the pressure in the channels 1 and 2 is controlled by the microfluidic droplet generation to obtain water-in-oil droplets.

7. A method for rapid detection of coliform bacteria according to claim 1 or 2, characterized in that, In step (4), the culture time is 3 to 5 hours.

8. A method for rapid detection of coliform bacteria according to claim 1 or 2, characterized in that, When the culture medium is EC-MUG medium, the maximum excitation wavelength is 355-375 nm and the maximum emission wavelength is 440-460 nm; When the culture medium is a mixture of EC medium and FDG, the maximum excitation wavelength is 490-492 nm and the maximum emission wavelength is 514-520 nm.

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