Method and device for detecting hydrogen sulfide yield of food-borne pathogenic bacteria

Abstract

The invention relates to the technical field of hydrogen sulfide detection, in particular to a method and device for detecting the yield of hydrogen sulfide of food-borne pathogenic bacteria. The device comprises a cell culture plate with a cover plate, and detection test paper and a sealing gasket are arranged between the cell culture plate and the cover plate. The hydrogen sulfide detection device provided by the invention is simple, and the required raw materials are easy to obtain; the requirement on sample treatment is low, and the sample can be loaded and measured only by carrying out proper liquid culture on the sample; according to the method, multiple (such as 96) samples can be detected at a time, the detection result is stable, the data uniformity is good, and the high-throughput detection requirement in scientific research and practical application can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogen sulfide detection, and in particular to a method and device for detecting the hydrogen sulfide production of foodborne pathogenic bacteria. Background Art

[0002] Hydrogen sulfide (H2S) is a colorless gas with a strong rotten egg smell, with a molecular weight of 34.08. It has a similar structure to water molecules, is slightly soluble in water, and has a density greater than air. As the third gas signaling molecule discovered after carbon monoxide (CO) and nitric oxide (NO), in recent years, more and more studies have found that endogenous H2S plays an important biological function in some physiological and pathological processes. Studies have found that H2S is closely related to protecting the cardiovascular system, cell energy metabolism, apoptosis, inflammation, cancer, and protecting cells from oxidative stress damage. In addition, some studies have found that at the whole organism level, H2S can extend the lifespan of nematodes and mice. In recent years, studies have found that most bacteria can also produce endogenous H2S. Similar to mammals, H2S plays an important role in bacteria. Most bacteria contain one or more H2S-producing enzymes, and these bacteria will release H2S when growing in rich medium or inorganic salt medium. The ability of bacteria to produce hydrogen sulfide is considered to be closely related to functions such as antibiotic resistance, biofilm formation, persister formation, and immune escape. Therefore, developing an efficient method to detect H2S in food, the environment, and living systems is of great significance for preventing food safety problems caused by foodborne pathogenic bacteria and studying their physiology and pathogenicity.

[0003] Currently, the commonly used methods for detecting hydrogen sulfide in foodborne pathogenic bacteria mainly include: colorimetric analysis, chromatography, fluorescence probe method, etc. Among them, colorimetric analysis has low cost and simple operation and is considered an ideal method. However, it has large errors and high requirements for the culture medium matrix, which is not conducive to detection in food matrices or the environment. Although chromatography has high stability and accuracy, its detection steps are very cumbersome, requiring special equipment and being very time-consuming; the fluorescence probe method is considered a promising tool in the field of life science research in recent years, but the preparation of fluorescence probes is very time-consuming and laborious, and the required reactants and reagents are often very expensive, unable to achieve the purpose of large-scale, rapid, and convenient detection. Therefore, there is an urgent need to develop a safe, stable, simple, and convenient method for detecting endogenous hydrogen sulfide in foodborne pathogenic bacteria. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a rapid, convenient, safe and stable method and device for detecting hydrogen sulfide produced by foodborne pathogenic bacteria. This method has low requirements for sample treatment, wide application range, safe and stable detection reagents, simple operation, and can be detected in large batches. The results are visible to the naked eye and can be converted for relative quantification. Non-professionals can also perform the detection.

[0005] In one aspect, the present invention provides a hydrogen sulfide detection device, which includes a cell culture plate with a cover plate, and a test paper and a gasket are arranged between the cell culture plate and the cover plate.

[0006] In some embodiments, the cell culture plate is a multi-well culture plate, such as 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, etc.

[0007] In some embodiments, the test paper is a filter paper adsorbed with a detection reagent that can detect H2S gas. The detection reagent is lead acetate. Preferably, the test paper is prepared by the following method: cut the filter paper, soak it in a 0.5% lead acetate solution, and then place it in an oven to dry to a constant weight.

[0008] In some embodiments, the thickness of the gasket is 0.2 mm.

[0009] In another aspect, the present invention provides a method for detecting the hydrogen sulfide production of foodborne pathogenic bacteria, including:

[0010] (1) Inoculate foodborne pathogenic bacteria in a culture medium and incubate overnight to obtain a bacterial culture solution in the stationary phase;

[0011] (2) Adjust the OD600 value of the bacterial culture solution to 0.1, and inoculate the diluted bacterial culture solution into the cell culture plate, 200 μL per well;

[0012] (3) Moisten the test paper with sterile water; after removing the excess water, place the test paper directly above the cell culture plate and closely fit it to each well;

[0013] (4) Cover the gasket directly above the test paper, and then cover the cover plate to make the periphery of the cell culture plate fit tightly without extra gaps;

[0014] (5) Place the sealed cell culture plate in a constant temperature shaker for cultivation;

[0015] (6) After the cultivation is completed, use a gel imager or a mobile phone to take pictures and record the depth of the black precipitate color on the surface of the test paper.

[0016] In some embodiments, the foodborne pathogenic bacteria include methicillin-resistant Staphylococcus aureus.

[0017] In some embodiments, in step (3), the test strip is prepared by the following method: Prepare a lead acetate solution containing 0.5%; Cut the filter paper (size: 125×85 mm); Fully immerse the filter paper in the lead acetate solution; Place the soaked filter paper in an oven and dry it to a constant weight; Seal and store it in a dry place.

[0018] Further, the filtration rate of the filter paper is 0 - 35 s, and the pore size is 80 - 120 μm.

[0019] In some embodiments, in step (5), during the cultivation process, L-cysteine is added to promote the release of endogenous hydrogen sulfide from foodborne pathogenic bacteria.

[0020] In some embodiments, in step (5), the speed of the shaker is controlled at 150 - 200 r / min.

[0021] In some embodiments, in step (6), ImageJ software is used for color conversion to avoid the influence of subjective factors, thereby realizing relative quantitative analysis.

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] 1. The hydrogen sulfide detection device provided by the present invention is simple, and the required raw materials are easily obtainable, only requiring lead acetate, rapid filter paper, and a cell culture plate, such as a 96-well plate;

[0024] 2. The hydrogen sulfide detection device and method provided by the present invention have low requirements for sample processing. Only appropriate liquid cultivation of the sample is required, and then it can be loaded for determination;

[0025] 3. The hydrogen sulfide detection device and method provided by the present invention can detect multiple (e.g., 96) samples at one time, and the detection results are stable, with good data homogeneity, and can meet the high-throughput detection requirements in scientific research and practical applications;

[0026] 4. The detection device and method provided by the present invention have a wide application range and can detect H2S produced by bacteria in fields such as food, environment, and medicine. The detection results are less limited by the types of bacteria;

[0027] 5. The hydrogen sulfide detection device provided by the present invention is simple to operate, and reading does not rely on instruments such as spectrophotometers and microplate readers;

[0028] 6. The hydrogen sulfide detection device and method provided by the present invention can quickly read data, the results are visible to the naked eye, and relative quantitative conversion can be performed. Non-professionals can also perform the detection. Description of the Drawings

[0029] The concept and specific structure of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features, and effects of the present invention.

[0030] Figure 1 Schematic diagram of the hydrogen sulfide detection device in the preferred embodiment of the present invention;

[0031] Figure 2 Photo of the test strip after detection in Example 1 of the present invention;

[0032] Figure 3 Shows the color conversion result of ImageJ software and the photo of the test strip after detection in Example 2 of the present invention. Detailed implementation manners

[0033] In order to make the purpose, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Example 1

[0035] Determination of the endogenous hydrogen sulfide production of methicillin-resistant Staphylococcus aureus under culture conditions of different concentrations of L-cysteine.

[0036] Using the methicillin-resistant Staphylococcus aureus standard strain ATCC BAA-1717 as the experimental strain, a series of concentration gradients of L-cysteine culture conditions were set to explore the effect of adding different concentrations of L-cysteine on the endogenous hydrogen sulfide production of methicillin-resistant Staphylococcus aureus.

[0037] First, dip the bacterial liquid stored at -80°C, streak-inoculate it onto tryptic soy agar (TSA) medium, and place it in a constant temperature and humidity incubator at 37°C for 12 h.

[0038] Pick a single colony from the TSA medium into tryptic soy broth (TSB), and place it in a shaker at 37°C and 180 r / min for 6 h.

[0039] Aspirate 1 mL of the bacterial liquid into a 1.5 mL centrifuge tube, centrifuge it at 8000 r / min for 1 min, discard the supernatant completely, add 0.85% normal saline to resuspend the bacteria, mix well and then perform the centrifugation operation (8000 r / min, 1 min), and repeat 3 times.

[0040] Add 1 mL of 0.85% normal saline to resuspend the bacterial cells, and adjust the OD600 value of the bacterial liquid to 1 for standby.

[0041] TSB medium was used as solvent to prepare TSB medium containing different concentration gradients of L-cysteine. The medium was mixed with the bacterial solution so that the final L-cysteine concentrations were 12800 / 6200 / 3200 μM, respectively.

[0042] Add the mixture of bacterial liquid and culture medium to a 96-well plate (set 12 parallel groups for each concentration, i.e. add to 12 wells), 200 μL per well; fully moisten the fully dried filter paper in sterile water; after removing excess water, place the test paper directly above the 96-well plate, fitting tightly to each test well. Cover the test paper directly above with a sealing pad, and then cover the 96-well plate with a lid, so that the wells are tightly fitted around the plate without any extra gaps;

[0043] The 96-well plate was fixed in a constant temperature shaker and cultured at 150 r / min for 12 h; photos were taken to record the changes in the black precipitate on the test strip.

[0044] The results are as follows Figure 2 As shown, the higher the L-cysteine concentration, the more hydrogen sulfide produced by methicillin-resistant Staphylococcus aureus, the darker the black precipitate on the test paper in the hydrogen sulfide detection device of the present invention, and the depth between the parallel groups is consistent, indicating that the hydrogen sulfide detection device of the present invention can relatively quantitatively detect the hydrogen sulfide gas production of foodborne pathogens, the results are stable, reliable and intuitive, and the operation is simple and convenient.

[0045] Example 2

[0046] Determination of the effect of glucose addition on endogenous hydrogen sulfide production by methicillin-resistant Staphylococcus aureus.

[0047] Taking the standard strain of methicillin-resistant Staphylococcus aureus ATCC BAA-1717 as the experimental strain, a series of glucose culture conditions with concentration gradients were set to explore the effect of adding different concentrations of glucose on the endogenous hydrogen sulfide production of methicillin-resistant Staphylococcus aureus.

[0048] First, dip the bacterial liquid frozen at -80°C, streak it onto tryptone soy agar (TSA) medium, and place it in a constant temperature and humidity incubator at 37°C for 12 hours.

[0049] A single colony on the TSA medium was picked up and placed in tryptone soy broth (TSB), and cultured in a shaker at 37°C and 180 rpm for 6 h.

[0050] Pipette 1 mL of bacterial solution into a 1.5 mL centrifuge tube, centrifuge at 8000 r / min for 1 min, discard the supernatant and add 0.85% saline to wash the bacteria, mix well and centrifuge again (8000 r / min, 1 min), repeat 3 times.

[0051] Resuspend the bacteria in 1 mL of 0.85% normal saline and adjust the OD600 value of the bacterial solution to 1 for standby.

[0052] Using TSB medium as a solvent, prepare TSB media containing different concentration gradients of glucose. Mix the media with the bacterial solution so that the final glucose concentrations are 0.5%, 0.45%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, 0.1%, 0.05%, 0 (wt / vol).

[0053] Add the mixture of the bacterial solution and the medium to a 96-well plate (set 4 parallel groups for each concentration, that is, add to 4 wells), 200 μL per well; fully moisten the filter paper after it is thoroughly dried in sterile water; after removing the excess water, place the test paper directly above the 96-well plate and closely fit it to each detection well. Cover the test paper with a gasket, and then cover the lid of the 96-well plate to make the periphery of the well plate fit tightly without extra gaps;

[0054] Fix the 96-well plate in a constant temperature shaker and culture it at 150 r / min for 12 h; take pictures to record the change of the black precipitate on the test paper.

[0055] Use ImageJ software to perform color conversion on the test results, so as to achieve relative quantitative analysis.

[0056] The results are as Figure 3 shown. As the glucose concentration increases, the hydrogen sulfide production of methicillin-resistant Staphylococcus aureus decreases instead. In the hydrogen sulfide detection device of the present invention, the degree of black discoloration on the test paper decreases, and the depth between parallel groups is consistent, indicating that combined with ImageJ software, the hydrogen sulfide detection device of the present invention can achieve relative quantitative analysis and has broad application prospects in scientific research and detection work.

Claims

1. A hydrogen sulfide detection device, the device comprising a cell culture plate with a cover plate, and a test paper and a gasket are provided between the cell culture plate and the cover plate.

2. The device according to claim 1, wherein The cell culture plate is a multi-well culture plate, having 6 wells, 12 wells, 24 wells, 48 wells, 96 wells or 384 wells.

3. The device according to claim 1, wherein The test paper is a filter paper adsorbed with a detection reagent, and the detection reagent is lead acetate.

4. The apparatus according to claim 1, wherein The thickness of the gasket is 0.2 mm.

5. A method for detecting the hydrogen sulfide production of foodborne pathogenic bacteria, comprising: (1) Inoculating foodborne pathogenic bacteria in a culture medium and incubating overnight to obtain a bacterial culture solution in the stationary phase; (2) Adjusting the OD600 value of the bacterial culture solution to 0.1, and inoculating the diluted bacterial culture solution into the cell culture plate, 200 μL per well; (3) Wetting the test paper with sterile water; after removing the excess water, placing the test paper directly above the cell culture plate and closely fitting it to each well; (4) Covering the gasket directly above the test paper, and then covering the cover plate to make the periphery of the cell culture plate closely fit without extra gaps; (5) Placing the sealed cell culture plate in a thermostatic shaker for culture; (6) After the culture is completed, using a gel imaging system or a mobile phone to take pictures and record the depth of the black precipitate color on the surface of the test paper.

6. The method according to claim 5, wherein, In step (3), the test paper is prepared by the following method: preparing a lead acetate solution containing 0.5%; cutting the filter paper; soaking the filter paper in the lead acetate solution; placing the soaked filter paper in an oven and drying it to constant weight; storing it sealed in a dry place.

7. The method according to claim 6, wherein, The filtration rate of the filter paper is 0 - 35 s, and the pore size is 80 - 120 μm.

8. The method according to claim 5, wherein, In step (5), during the culture process, L-cysteine is added.

9. The method according to claim 5, wherein, In step (5), the speed of the shaker is controlled at 150 - 200 r / min.

10. The method according to claim 5, wherein In step (6), ImageJ software is used for color conversion to achieve relative quantitative analysis.

Images