A method for evaluating bacterial activity based on GelRed dye

Abstract

The present invention discloses a method for evaluating bacterial activity based on GelRed dye, wherein metal ions are first mixed with bacterial samples, and then allowed to stand for a certain period of time to obtain a mixed bacterial solution, and then GelRed dye is added to the mixed bacterial solution to mix evenly and then allowed to stand for a certain period of time; ultrapure water is added for centrifugation, the supernatant is discarded, and then Syto9 solution is added for mixing; finally, a fluorescence photometer is used for detection. The evaluation method of the present invention has the characteristics of high sensitivity, rapidity and low toxicity.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial activity evaluation, and in particular to a bacterial activity evaluation method based on GelRed dye. Background Art

[0002] Bacterial activity assessment plays an important role in the fields of bacterial detection and antimicrobial drug development. The plate count method is considered to be the standard method for evaluating bacterial activity. This method evenly distributes bacteria on a solid culture medium by coating. Single bacteria reproduce by division and eventually form dispersed colonies visible to the naked eye on the solid culture medium. The plate count method requires a lot of equipment, reagents and time. In addition, colony counts may be affected by different culture conditions, and temperature and cell growth rate may increase random errors.

[0003] In the past two decades, scientists have been trying to develop methods to quickly and efficiently distinguish between dead and alive bacteria, including electron microscopy, real-time fluorescence quantitative PCR (qPCR), surface-enhanced Raman spectroscopy, aggregation-induced emission (AIE), fluorescence staining combined with fluorescence spectroscopy, optical microscopy or flow cytometry (FCM). These technologies are difficult to meet the requirements of high sensitivity and rapid detection at the same time. For example, fluorescence spectroscopy is difficult to accurately determine the bacterial mortality rate after the action of antibiotics. As a rapid and high-throughput method, FCM is difficult to detect bacterial mortality rates below 10%. Although the recently developed high-sensitivity FCM can reduce this detection limit to 2%-5%, it is still difficult to meet the requirements of sensitivity and rapid detection.

[0004] At the same time, various fluorescent dyes are also used to improve the sensitivity of identifying live / dead bacteria, such as propidium iodide (PI), diacetate and rhodamine. Among them, PI is the most widely used dye. PI only penetrates damaged bacterial membranes and stains DNA with red fluorescence to achieve the effect of selectively staining dead cells. However, PI has problems of high background noise and low sensitivity, and is a toxic reagent to mammalian cells.

[0005] Currently, safe nucleic acid dyes such as GelRed, SYBRsafe and SYTO 9 have been developed and widely used in DNA gel electrophoresis. Compared with PI dye, GelRed has the characteristics of high sensitivity and low toxicity. There are no reports on the use of GelRed for bacterial staining.

[0006] In summary, there is an urgent need for a method that can detect bacterial activity with low toxicity, high efficiency and high sensitivity. Summary of the invention

[0007] In order to overcome the above-mentioned defects of the prior art, a bacterial activity assessment method based on GelRed dye is provided, which can quickly, efficiently and highly sensitively distinguish between dead and alive bacteria, and the materials used are low in toxicity, high in safety and simple in detection operation.

[0008] The technical solution adopted to achieve the purpose of the present invention is:

[0009] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0010] (1) After mixing the metal ions and the bacterial sample, the mixture is allowed to stand for a certain period of time to obtain a mixed bacterial solution;

[0011] (2) Add GelRed dye to the mixed bacterial solution, mix well, and let stand for a certain period of time;

[0012] (3) adding ultrapure water to the mixed bacterial solution of step (2), centrifuging, discarding the supernatant, and then adding the syto9 solution to mix;

[0013] (4) The mixed bacterial solution of step (3) is detected using a fluorescence photometer.

[0014] Preferably, in step (1), the metal ion is Mg 2+ , and its final concentration is above 10mM.

[0015] Preferably, in step (1), the standing time is 10-15 minutes.

[0016] Preferably, in step (2), the standing time is 20-30 minutes.

[0017] Preferably, in step (2), the concentration of the GelRed dye is 0.1 μg mL -1 above.

[0018] Preferably, in step (3), ultrapure water is added and centrifuged at 8000 g for 5-8 minutes.

[0019] Preferably, in step (3), the final concentration of the syto9 solution is above 1 μM.

[0020] The present invention also provides an application of a bacterial activity evaluation method based on GelRed dye in the field of bacterial detection, wherein the application contains the above-mentioned bacterial activity evaluation method based on GelRed dye.

[0021] The present invention further provides an application of a bacterial activity evaluation method based on GelRed dye in the field of antibacterial drug development, wherein the application contains the above-mentioned bacterial activity evaluation method based on GelRed dye.

[0022] By adopting the above technical solution, the present invention has the following technical effects and advantages:

[0023] (1) High sensitivity: The present invention uses high-concentration metal ions to mix and react with bacterial samples for a period of time, thereby reducing the adsorption of dyes on the bacterial surface and reducing background noise. The fluorescence ratio method established by combining GelRed dye and SYTO 9 can effectively detect low-ratio dead bacteria samples and can detect 0.1% dead bacteria, which is 2 orders of magnitude higher than the detection limit of 2%-5% of high-sensitivity FCM.

[0024] (2) Rapidity: The method comprises mixing a bacterial sample with a high concentration of metal ions, adding GelRed dye for reaction, adding ultrapure water for centrifugation, adding Syto9 solution for mixing, and using a fluorescence photometer for detection. The operation steps are simple and rapid, and the requirements for the experimenter are low.

[0025] (2) Low toxicity: GelRed dye is a highly sensitive, low-toxic and ultra-stable fluorescent nucleic acid gel staining reagent. Its water-soluble dye has been certified as safe by the U.S. Environmental Protection Agency. The waste can be directly poured into the sewer without causing any environmental pollution. GelRed dye is less harmful to mammals than PI and is a relatively safe dye, which reduces the harm of the dye to experimental personnel. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure is a schematic diagram of a process of evaluating bacterial activity based on GelRed dye according to the present invention.

[0027] Figure 2 The fluorescence spectra of live and dead bacteria after staining with different concentrations of GelRed in Examples 1-5. (ae correspond to the fluorescence spectra of dead and live bacteria in Examples 1-5 with a GelRed concentration of 0.02-2ug / mL; f is the ratio of dead and live bacteria at different GelRed concentrations at 620nm).

[0028] Figure 3 Figure 1 is the fluorescence spectra of live and dead bacteria after GelRed staining in the presence of different concentrations of MgCl2. (ae) Corresponding to Examples 6-10, bacteria were stained with 1ug / mL GelRed in the presence of different concentrations of MgCl2, and the MgCl2 concentration range was 0.0005-500mM. The left figure is a representative bacterial solution under UV light after staining. (f) is a comparison of the FL ratio of dead and live bacteria at 620nm under different MgCl2 concentrations.

[0029] Figure 4The ratio fluorescence method for detecting dead and live bacteria in Example 9. (a) FL intensity of dead bacteria at 620 nm after GelRed staining, with a bacterial concentration of 0.05 to 10×10 9 cfu / mL. (b) Relationship between dead bacteria concentration and red FL intensity. (c) FL intensity of live bacteria at 620nm after GelRed staining. (d) Fluorescence spectra of live and dead bacteria after GelRed / SYTO 9 double staining, with dead bacteria ratio ranging from 0.1% to 100%. (e) Comparison of fluorescence intensity at 500nm and 620nm. (f) Relationship between dead bacteria ratio and ratio fluorescence (FL620nm / 500nm). Data are expressed as mean±SD (n=3).

[0030] Figure 5 Microscopic observation and flow cytometric analysis of dead / live bacteria in Example 11. (ae) Representative fluorescence images of SYTO 9 / GelRed double-stained bacteria. The proportion of dead bacteria is 0% (a), 20% (b), 50% (c), 80% (d), and 100% (e). (a1-e1) Green fluorescence of SYTO 9 staining. (a2-e2) Red fluorescence for GelRed staining. (fi) Flow cytometric histogram of bacteria double-stained with SYTO9 / GelRed; the proportion of dead bacteria is 0% (f), 20% (f), 80% (h), and 100% (i); (f1-i1) Green fluorescence distribution of SYTO 9 staining; (f2-i2) Red fluorescence distribution of GelRed sta. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The present invention reduces the adsorption of dyes on bacterial surfaces by using high-concentration metal ions, thereby reducing background noise, and uses a safer dye, GelRed, instead of PI, to reduce the possibility of harm to experimenters caused by the dyes.

[0033] At present, syto9 / PI live and dead bacteria staining is a widely used method in the market to detect the live and dead of bacteria. Since PI itself is toxic to mammals and has high background noise in staining, it is difficult to detect samples with a low percentage of dead bacteria to a certain extent.

[0034] Through a large number of experimental studies, the inventor accidentally discovered when trying to use GelRed to stain bacteria: GelRed can only penetrate dead bacteria, not live bacteria; this principle can be used to identify dead and live bacteria. In addition, it was found through research that the cell walls of live bacteria will adsorb GelRed molecules, resulting in non-specific staining. 2+ ) can remove non-specific adsorption and increase the fluorescence staining contrast between dead and live bacteria.

[0035] Based on this, the present invention proposes a method for evaluating bacterial activity based on GelRed dye.

[0036] like Figure 1 As shown, a method for evaluating bacterial activity based on GelRed dye comprises the following steps:

[0037] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration 10 mM) and let stand for 10 minutes;

[0038] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 0.1 μg mL -1 ), mix well and let stand for 20 minutes;

[0039] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0040] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0041] Preferably, Mg 2+ Ca 2+ , K + 、Na + 、Zn 2+ 、Al 3+ , Fe 3+ The non-specific adsorption of GelRed dye on live bacteria can be effectively removed by replacing inorganic salts such as chlorinated salts, thereby greatly improving the selectivity of GelRed dye for dead bacteria.

[0042] This method uses bacterial samples and Mg 2+ Mixing and combining GelRed dye and syto9 solution can effectively improve the sensitivity of detection and reduce background noise. The low toxicity of GelRed dye can reduce the harm of the dye to the human body.

[0043] Example 1

[0044] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0045] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration 10 mM) and let stand for 10 minutes;

[0046] (2) Continue to mix evenly and let stand for 20 minutes;

[0047] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0048] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0049] Example 2

[0050] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0051] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration 10 mM) and let stand for 10 minutes;

[0052] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 0.02 μg mL -1 ), mix well and let stand for 20 minutes;

[0053] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0054] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0055] Example 3

[0056] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0057] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration 10 mM) and let stand for 10 minutes;

[0058] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 0.1 μg mL -1 ), mix well and let stand for 20 minutes;

[0059] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0060] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0061] Example 4

[0062] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0063] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration 10 mM) and let stand for 10 minutes;

[0064] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 0.5 μg mL -1 ), mix well and let stand for 20 minutes;

[0065] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0066] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0067] Example 5

[0068] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0069] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration 10 mM) and let stand for 10 minutes;

[0070] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 2 μg mL -1 ), mix well and let stand for 20 minutes;

[0071] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0072] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0073] Example 6

[0074] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0075] (1) Let the bacterial sample stand for 10 minutes;

[0076] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 1 μg mL -1 ), mix well and let stand for 20 minutes;

[0077] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0078] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0079] Example 7

[0080] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0081] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration reach 0.005 mM) and let stand for 10 minutes;

[0082] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 1 μg mL -1 ), mix well and let stand for 20 minutes;

[0083] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0084] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0085] Example 8

[0086] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0087] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration 0.5 mM) and let stand for 10 minutes;

[0088] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 1 μg mL -1 ), mix well and let stand for 20 minutes;

[0089] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0090] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0091] Example 9

[0092] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0093] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to make the final concentration 10 mM) and let stand for 10 minutes;

[0094] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 1 μg mL -1 ), mix well and let stand for 20 minutes;

[0095] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0096] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0097] Example 10

[0098] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0099] (1) First, mix the bacterial sample with 100 mM Mg 2+ Mix (to a final concentration of 100 mM) and let stand for 10 minutes;

[0100] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 1 μg mL -1 ), mix well and let stand for 20 minutes;

[0101] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0102] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0103] Embodiment 11

[0104] A method for evaluating bacterial activity based on GelRed dye, comprising the following steps:

[0105] (1) Bacterial samples with a dead bacteria ratio of 0%, 20%, 50%, 80%, and 100% were mixed with 100 mM Mg 2+ Mix (to make the final concentration 10 mM) and let stand for 10 minutes;

[0106] (2) Add 20 μg mL -1 GelRed dye (to a final concentration of 1 μg mL -1 ), mix well and let stand for 20 minutes;

[0107] (3) Add ultrapure water and centrifuge at 8000 g for 5 min, discard the supernatant, wash twice, add 10 μM syto9 solution (to a final concentration of 1 μM) and mix well;

[0108] (4) The mixed bacterial solution in step (3) is detected using a fluorescence photometer.

[0109] Experimental Results

[0110] Figure 2 The fluorescence spectra of live and dead bacteria after dyeing with different concentrations of GelRed in Examples 1 to 5 are shown. It can be seen from the spectra that 0.1ug / mL GelRed dye has the best effect on distinguishing dead and live bacteria, but the background noise of GelRed dyeing live bacteria is relatively high, and the nonspecific adsorption of the live bacteria surface to the dye needs to be reduced.

[0111] Figure 3 It is shown that after adding different concentrations of MgCl2 solution in Examples 6-10, the non-specific adsorption of GelRed dye by live bacteria is reduced. The final concentration of MgCl2 is 10mM. 2+ The spectrum of shows that there is basically no absorption peak of GelRed dye for live bacteria, and the absorption peak of GelRed dye for dead bacteria has the smallest impact. This concentration has the best effect on distinguishing between live and dead bacteria.

[0112] Figure 4The sensitivity of the ratio fluorescence method of Example 9 for detecting dead and live bacteria is shown. As can be seen from Figures ac, the red fluorescence increases with the increase in the concentration of dead bacteria, and basically presents a linear relationship; while the red fluorescence intensity of live bacteria remains basically unchanged. Considering that single-wavelength emission is affected by many factors, such as temperature, excitation wavelength, and instrument type, etc. In contrast, the ratio fluorescence method can be self-calibrated by two dependent emissions, and has better repeatability and sensitivity than intensity-based methods. Therefore, we use another safer dye SYTO 9 as a reference to establish the ratio fluorescence method. This method is suitable for detecting a small amount of dead bacteria from a mixture of live / dead cells. SYTO 9 is a green fluorescent dye that can penetrate the cell membrane and can mark all bacteria as green regardless of the membrane state of the bacteria. The fluorescence spectra and fluorescence intensities of dead / live bacterial mixtures of different ratios are shown in Figures df (500nm:SYTO 9; 620nm:GelRed). As the dead bacteria ratio increases, the green filter rate decreases and the red filter rate increases. For dead bacteria, both GelRed and SYTO can penetrate the cells, but GelRed appears to bind dsDNA more strongly than SYTO 9, resulting in a high ratiometric fluorescence signal.

[0113] Figure 5 The dead / live bacteria microscopic observation and flow cytometric analysis of Example 11, Figures a1-e1 and a2-e2 are fluorescence microscopic images of GelRed / SYTO 9 double-stained bacteria. Since GelRed replaces SYTO 9, live bacteria show green fluorescence, while dead bacteria only show red fluorescence, which is consistent with the FL spectrum analysis results ( Figure 4 ). Figures f1-i1 show the flow cytometric histograms of green FL and red FL in standard solutions of live / dead bacteria mixtures containing 0%, 20%, 80%, and 100% dead bacteria. The proportion defined as dead bacteria in the right section of Figure f2-i2 increases with the increase of the theoretical dead bacteria ratio. The average green fluorescence decreases with the increase of red fluorescence, further confirming that GelRed and SYTO 9 are competitive in binding to dead bacteria dsDNA. The results show that the number of dead bacteria detected has a good correlation with the theoretical value.

[0114] The above results show that we found that live bacteria can exclude GelRed dye, however, it can easily penetrate dead bacteria and be directly absorbed onto bacterial DNA. Some inorganic salts such as MgCl2 and CaCl2 can effectively remove the nonspecific adsorption of GelRed dye on live bacteria, thereby greatly improving the selectivity of GelRed dye for dead bacteria. We combined the membrane permeable SYTO 9 to establish an efficient and highly sensitive fluorescence detection method for quantifying bacterial viability. This method has the characteristics of high safety, high sensitivity, and fast detection speed, and it has many potential applications in microbial research and antimicrobial drug development in the future.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for evaluating bacterial activity based on GelRed dye, characterized in that: The following steps are involved: (1) After mixing the metal ions and the bacterial sample, the mixture is allowed to stand for a certain period of time to obtain a mixed bacterial solution; (2) Add GelRed dye to the mixed bacterial solution, mix well, and let stand for a certain period of time; (3) adding ultrapure water to the mixed bacterial solution of step (2), centrifuging, discarding the supernatant, and then adding the syto9 solution to mix; (4) detecting the mixed bacterial solution of step (3) using a fluorescence photometer; In step (1), the metal ion is Mg 2+ .

2. A method for evaluating bacterial activity based on GelRed dye according to claim 1, characterized in that: The Mg 2 + The final concentration is above 10 mM.

3. A method for evaluating bacterial activity based on GelRed dye according to claim 1, characterized in that: In step (1), the standing time is 10-15 minutes.

4. A method for evaluating bacterial activity based on GelRed dye according to claim 1, characterized in that: In step (2), the standing time is 20-30 minutes.

5. The method for evaluating bacterial activity based on GelRed dye according to claim 1, characterized in that: In step (2), the concentration of the GelRed dye is 0.1 μg mL -1 above.

6. The method for evaluating bacterial activity based on GelRed dye according to claim 1, characterized in that: In step (3), add ultrapure water and centrifuge at 8000 g for 5-8 minutes.

7. A method for evaluating bacterial activity based on GelRed dye according to claim 1, characterized in that: In step (3), the final concentration of the syto9 solution is greater than 1 μM.

8. Application of a bacterial activity assessment method based on GelRed dye in the field of bacterial detection, characterized in that: The application comprises the bacterial activity assessment method based on GelRed dye as claimed in claim 1.

9. Application of a bacterial activity evaluation method based on GelRed dye in the field of antibacterial drug development, characterized in that: The application comprises the bacterial activity assessment method based on GelRed dye as claimed in claim 1.

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