Method for rapidly detecting activity of environment-friendly fungicide

Abstract

The invention discloses a method for rapidly detecting the activity of an environment-friendly fungicide. Specifically, the metabolic capability of a specific substrate is monitored and evaluated according to the characteristics of color change after tetrazole violet obtains electrons. The correlation between the bacterial activity and the change of the light absorption signal of tetrazole purple under 570nm is obtained by measuring the change of the light absorption signal of the bacteria under the condition of the tetrazole purple indicator. And establishing a standard curve of the tetrazole violet absorbance and the bacterial activity. And detecting the change of a bacterial absorption light signal under the tetrazole violet indicator of the environment-friendly microbial agent, and comparing the change with the standard curve to quickly obtain the activity of the environment-friendly microbial agent. The method for rapidly detecting the activity of the environment-friendly microbial agent has the advantages of simplicity, convenience, simplicity in operation, low toxicity, continuous monitoring and accuracy in multi-bacterium detection, and is an efficient solution for detecting the activity of the bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an activity detection method for an environmentally friendly bacterial agent based on tetrazolium violet. Background Art

[0002] Environmentally friendly microbial agents can be used in the bio-enhancement process in the fields of sewage treatment, soil remediation, etc. The methods for detecting the activity of microbial agents include colony counting method and protein determination method. Plate counting method can only detect the number of live bacteria, the detection time lasts for several days, and can only detect less than 1% of culturable microorganisms in the natural environment. The new fluorescent staining method, enzyme-linked immunosorbent assay, flow cytometry and other methods have complex operation processes, high technical and equipment requirements, and are not conducive to on-site use; in addition, the detection process is also prone to damage to bacteria. Continuous monitoring cannot be achieved.

[0003] At present, the commonly used biological agent activity detection methods in agriculture, food and other industries have problems such as slow activity detection speed and efficiency, and large interference from pollutants during their application in the environmental protection industry. After the environmental protection agent is added to polluted water and soil, its activity will be greatly affected due to the complexity of toxic substances, environmental factors, etc., and the current common activity detection methods cannot achieve rapid detection of the impact of pollutants on the activity of the agent. The development of a simple, low-toxic and non-destructive method for detecting the activity of the agent has important application value. In view of this, the present invention provides a method for rapid detection of the activity of environmental protection agents, so as to accurately measure the bacterial activity of environmental protection agents, while reducing damage to bacteria and achieving long-term monitoring. Summary of the invention

[0004] In view of the problems existing in the existing microbial activity detection technology, such as slow activity detection speed and efficiency, large interference from pollutants, etc., the present invention provides a detection method with high efficiency, rapidity and strong anti-interference ability for the activity detection of environmentally friendly microbial agents. Specifically, the metabolic capacity of a specific substrate is monitored and evaluated by the color change characteristics of tetrazolium violet after it obtains electrons. Microorganisms oxidize the absorbed substrates through respiration to obtain energy, and the electrons generated in the process are easily absorbed by tetrazolium violet, thereby indicating the process and degree of bacterial utilization of the substrate. After tetrazolium violet obtains electrons, it gradually changes from colorless to pink, purple, red, blue-purple and blue; the more electrons it obtains, the darker the color. Its determination process requires an enzyme marker or a spectrophotometer, which can complete the measurement in a short time, and can also be used for long-term monitoring of changes in the activity of bacterial solutions.

[0005] The purpose of the present invention is achieved by at least one of the following technical solutions:

[0006] A method for rapid detection of the activity of an environmentally friendly bacterial agent comprises the following steps:

[0007] 1) A series of bacterial solutions with the same total bacterial concentration and different activities are obtained, and a tetrazolium violet indicator is added to measure the light absorption signal at 570 nm to obtain a series of absorbance values ​​under different bacterial activities.

[0008] 2) Establish a standard curve between absorbance value and cell activity;

[0009] 3) The environmentally friendly bacterial agent to be tested is operated according to step 1), the absorbance value is determined, the absorbance is obtained by data processing related to the absorbance value, and the bacterial activity in the environmentally friendly bacterial agent to be tested is calculated according to the standard curve.

[0010] Preferably, the cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas stutzeri, Rhodococcus erythroxysporum, or Halomonas.

[0011] Furthermore, the bacterial solutions with the same total bacterial concentration and different activities in step 1) were inoculated in liquid LB medium and 70% isopropanol solution, respectively. After culturing for 12 hours, the supernatant was discarded by centrifugation, and the precipitated bacteria were washed 3 times with PBS solution to obtain live bacteria / dead bacteria solutions; the solutions were diluted to a total bacterial concentration of 10 5 cells / mL;

[0012] Furthermore, by mixing live bacteria and dead bacteria solutions in different proportions, the activity gradient was set to 0%, 10%, 20%, 40%, 60%, 80%, and 100%, and standard bacterial solutions with the same total bacterial concentration and different activities were obtained;

[0013] Furthermore, a series of standard solutions with different concentrations were added with tetrazolium violet indicator to make the concentration reach 0.01%, and after being placed in an incubator for 10 minutes, the absorbance at 570 nm was measured to obtain a corresponding series of absorbance values; the above steps were repeated twice, and the three sets of data obtained were linearly fitted to establish a standard curve of Escherichia coli bacterial activity and absorbance value;

[0014] Further, the culture bottle was sterilized at 121°C, and the environmentally friendly bacterial agent was inoculated in the culture bottle containing LB liquid culture medium according to the instructions, and cultured at 25-35°C and 150-300rpm for 12-48h to obtain the logarithmic phase bacterial colony. The measurement process was kept at a constant temperature.

[0015] Furthermore, the absorbance value is obtained according to step 3), and the activity of the bacterial agent is obtained by corresponding it according to the standard curve of bacterial activity. The absorbance value of tetrazolium violet at 570nm is a=K*V+b, wherein V is the normalized bacterial activity, K is the proportional coefficient determined by the bacterial species and size, and b is the reference value. In the process of measuring the activity of the bacterial agent, the absorbance value a of the bacterial solution after adding tetrazolium violet is first detected by an ELISA / spectrophotometer, and then the bacterial activity is obtained by V=(ab) / k×100%.

[0016] Compared with the prior art, the advantages of the technical solution of the present invention are:

[0017] The present invention uses tetrazolium violet to indicate the activity of environmentally friendly bacterial agents for the first time, and monitors and evaluates the metabolic capacity of specific substrates through the characteristics of color changes after tetrazolium violet obtains electrons. Microorganisms oxidize the absorbed substrates to obtain energy through respiration, and the electrons generated in the process are easily absorbed by tetrazolium violet, thereby indicating the process and degree of bacteria using the substrate. After tetrazolium violet obtains electrons, it gradually changes from colorless to pink, purple, red, blue-purple and blue; the more electrons it obtains, the darker the color. The present invention establishes a method for detecting the cell activity of environmentally friendly bacterial agents using an enzyme marker / spectrophotometer. Compared with the existing plate counting method, fluorescent staining method, enzyme-linked immunosorbent assay, etc., the method has low cost, fast detection speed, simple operation, and the detection process does not harm bacteria, and has great technical advantages. The method of the present invention is simple to operate, and only requires a culture bottle and an enzyme marker / spectrophotometer to achieve it. It does not require a complicated culture process and reagent preparation process, and the test can be completed in a short time, saving manpower, material resources and time. Can be used for low-toxicity continuous monitoring: Tetrazolium violet is an indicator commonly used in biolog phenotypic microarray technology. It is an indicator often used for continuous monitoring of cell activity. It has low toxicity and is less harmful to bacterial activity. It is a low-toxic indicator suitable for long-term monitoring and can reflect the growth and death of bacteria in real time. The cell activity established based on tetrazolium violet and cell active substances can be used to characterize the activity of bacterial flora in a variety of complex environmental conditions and toxic substances. Its accuracy is much higher than other methods, which can greatly improve the activity detection standards of environmentally friendly bacterial agents.

[0018] In summary, the method for rapid detection of environmentally friendly bacterial agent activity provided by the present invention is simple in method, easy to operate, low in toxicity and realizes continuous monitoring, and at the same time has the accuracy of multi-bacteria detection. It is a new solution for efficient bacterial activity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The standard curve is based on the autocorrelation of cell activity and absorbance at 570 nm established for E. coli.

[0020] Figure 2The activity of commercial bacterial agents is obtained by comparing with the standard curve of Escherichia coli cell activity.

[0021] Figure 3 The figure is an autocorrelation standard curve of cell activity-570nm absorbance established based on Corynebacterium glutamicum. DETAILED DESCRIPTION

[0022] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention. It should be noted that any process not described in detail below can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0023] Example 1: Construction of a standard curve based on Escherichia coli

[0024] 1. Calibration of standard curve: Escherichia coli (Escherichia coli, deposited in China Industrial Microbiological Culture Collection Center, deposit number CICC 10899) was inoculated in liquid LB medium and 70% isopropanol solution, respectively. After culturing for 12 hours, the supernatant was discarded by centrifugation, and the precipitated bacteria were washed three times with PBS solution to obtain live bacteria / dead bacteria solution; the live bacteria / dead bacteria solution was diluted to a total bacterial concentration of 10 5 cells / mL;

[0025] 2. Mix live bacteria and dead bacteria solutions in different proportions, set the activity gradient to 0%, 10%, 20%, 40%, 60%, 80%, 100%, and obtain standard bacterial solutions with the same total bacterial concentration and different activities;

[0026] 3. Add tetrazolium violet indicator to a series of standard solutions with different concentrations to make the concentration reach 0.01%, place them in an incubator and incubate them for 10 minutes, then measure the absorbance at 570 nm to obtain a series of corresponding absorbance values;

[0027] 4. Repeat the above steps twice, perform linear fitting on the three sets of data obtained, and establish a standard curve of E. coli bacterial activity and absorbance value, such as Figure 1 .

[0028] 5. Measurement of bacterial agent samples: Take a commercial bacterial agent (Biwofeng SF bacterial agent) for testing, where the effective live bacteria count is ≥ 200 million / g. It is mainly screened from nature and can be used to treat chemical wastewater containing components such as surfactants, dyes, coatings and benzene. Take 5g of the bacterial agent and add it to 100mL of water and stir to activate it. Under sterile conditions, inoculate the bacterial agent in LB liquid culture medium and culture it for 24 to 48 hours to obtain a mixed bacterial solution in the logarithmic phase. According to the methods of steps 1 and 2, a mixed bacterial solution with different activity gradients is obtained, and then the absorbance value is obtained as described in step 3; then the absorbance value is compared with the standard curve to obtain the activity of the mixed bacterial community. Repeat the above steps twice to obtain parallelism and ensure the stability of the results. Figure 2 . According to the formula, the following Table 1 is obtained.

[0029] Table 1

[0030] Actual activity of environmentally friendly microbial agents Environmentally friendly bacterial agent activity measurement 0％ (1.20±0.14)％ 10％ (12.30±1.32)％ 20％ (22.10±1.94)％ 40％ (41.71±2.48)％ 60％ (60.95±1.39)％ 80％ (82.46±2.96)％ 100％ (108.85±6.41)％

[0031] The results in Table 1 show that the method can be used to detect bacterial activity. It only needs to establish a standard curve and add an indicator to achieve rapid measurement. The operation is easy and the accuracy is high. Low concentrations of tetrazolium violet cause little damage to bacteria and can be used to detect changes in bacterial activity for a long time.

[0032] Example 2: Construction of a standard curve based on Corynebacterium glutamicum

[0033] 1. Calibration of standard curve: Corynebacterium glutamicum (deposited in China Industrial Microbiological Culture Collection Center, deposit number CICC 20192) was inoculated in liquid LB medium and 70% isopropanol solution, respectively. After culturing for 12 hours, the supernatant was discarded by centrifugation, and the precipitated bacteria were washed three times with PBS solution to obtain live bacteria / dead bacteria solution; the solution was diluted to a total bacterial concentration of 10 5 cells / mL;

[0034] 2. Mix live bacteria and dead bacteria solutions in different proportions, set the activity gradient to 0%, 10%, 20%, 40%, 60%, 80%, 100%, and obtain standard bacterial solutions with the same total bacterial concentration and different activities;

[0035] 3. Add tetrazolium violet indicator to a series of standard solutions with different concentrations to make the concentration reach 0.01%, place them in an incubator and incubate them for 10 minutes, then measure the absorbance at 570 nm to obtain a series of corresponding absorbance values;

[0036] 4. Repeat the above steps twice, perform linear fitting on the three sets of data obtained, and establish a standard curve of Corynebacterium glutamicum activity and absorbance value, such as Figure 3 .

[0037] 5. Obtain the absorbance value of the commercial bacterial agent according to the method in Example 1; then compare the absorbance value with the standard curve to obtain the activity of the mixed bacterial community. Repeat the above steps twice to obtain parallelism and ensure the stability of the results. Finally, convert according to the formula to obtain the following Table 2.

[0038] Table 2

[0039] Actual activity of environmentally friendly microbial agents Environmentally friendly bacterial agent activity measurement 0％ (2.51±0.13)％ 10％ (12.85±1.23)％ 20％ (21.97±0.87)％ 40％ (40.23±2.31)％ 60％ (58.15±1.30)％ 80％ (78.18±2.76)％ 100％ (102.74±5.97)％

[0040] The results in Table 2 show that when this method is used to detect bacterial activity, the standard curve selects commonly used bacterial species, and its error results do not affect the determination of bacterial agent activity.

[0041] The above are only preferred implementation strategies of the present invention. It should be noted that the above preferred implementations should not be regarded as limiting the present invention. The scope of protection of the present invention shall be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A rapid detection method for the activity of an environmentally friendly bacterial agent, characterized in that: The following steps are involved: (1) Select standard bacterial solutions with different bacterial activities but the same total bacterial concentration, add tetrazolium violet indicator to measure the light absorption signal at 570 nm, and obtain the absorbance values ​​under different bacterial activities by data processing related to the absorbance values; (2) Establish a standard curve between tetrazolium violet absorbance and bacterial activity; (3) The environmentally friendly bacterial agent to be tested is operated according to step (1), the absorbance value is measured, the absorbance is obtained by data processing related to the absorbance value, and the bacterial activity in the environmentally friendly bacterial agent to be tested is calculated according to the standard curve.

2. The rapid detection method for the activity of environmentally friendly bacterial agents according to claim 1, characterized in that: Step (1) The bacterial solutions with the same total bacterial concentration and different activities of the standard bacterial solution were inoculated into liquid LB medium and 70% isopropanol solution, respectively. After culturing for 12 hours, the supernatant was discarded by centrifugation, and the precipitated bacteria were washed three times with PBS solution to obtain live bacteria / dead bacteria solutions; the solutions were diluted to a total bacterial concentration of 10 5 cells / ml for later use.

3. The rapid detection method for the activity of environmentally friendly bacterial agents according to claim 2, characterized in that: The absorbance determination is carried out using an ELISA / spectrophotometer to measure the light absorption signals at 570 nm of a series of standard bacterial solutions with different bacterial activities but the same total bacterial concentration.

4. The rapid detection method for the activity of environmentally friendly bacterial agents according to claim 2, characterized in that: By mixing live and dead bacteria solutions in different proportions, the activity gradient was set to 0%, 10%, 20%, 40%, 60%, 80%, and 100% to obtain standard bacterial solutions with the same total bacterial concentration but different activities.

5. The rapid detection method for the activity of environmentally friendly bacterial agents according to claim 4, characterized in that: A series of standard solutions of different concentrations were added with tetrazolium violet indicator to make the concentration reach 0.01%, and then placed in an incubator for 8-12 minutes (specifically 10 minutes), and the absorbance at 570 nm was measured to obtain a corresponding series of absorbance values; the above steps were repeated twice, and the three sets of data were linearly fitted to establish a standard curve of bacterial activity and absorbance value.

6. The rapid detection method for the activity of environmentally friendly bacterial agents according to claim 1, characterized in that: The standard curve of tetrazolium violet absorbance and bacterial activity described in step (2) is normalized to establish a standard curve of tetrazolium violet absorbance and bacterial activity; In the culture process described in step (3), the culture bottle is first sterilized at 121°C with high temperature and wet heat, and the environmentally friendly bacterial agent is inoculated in the culture bottle containing LB liquid culture medium according to the instructions, and cultured at 25-35°C and 150-300rpm for 24-48h to obtain a logarithmic phase bacterial colony; the measurement process is maintained at a constant temperature.

7. The rapid detection method for the activity of an environmentally friendly bacterial agent according to claim 1, characterized in that: Step (3) measures the absorbance value obtained, and uses the absorbance of tetrazolium violet and the standard curve of bacterial activity to obtain the activity of the bacterial agent.

8. The rapid detection method for the activity of the environmentally friendly bacterial agent according to claim 7, characterized in that: The absorbance value of tetrazolium violet at 570nm is a=K*V+b, where V is the normalized bacterial activity, K is the proportional coefficient determined by the bacterial species and size, and b is the reference value.

9. The rapid detection method for the activity of an environmentally friendly bacterial agent according to claim 7, characterized in that: In the process of measuring the activity of the bacterial agent, the absorbance value a is first detected by an ELISA reader / spectrophotometer, and then the bacterial activity is obtained by V=1 / k(ab)×100%.

10. The rapid detection method for the activity of an environmentally friendly bacterial agent according to any one of claims 1 to 9, characterized in that: The standard bacteria are Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas stutzeri, Rhodococcus erythroxysporum, and Halomonas.

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