A quantitative detection method for cell membrane permeability Δ conductivity and conductivity detector

Abstract

The present invention relates to the technical field of cell membrane permeability detection, and specifically to a method for quantitative detection of cell membrane permeability Δ conductivity. By studying the cell membrane permeability of the first cotyledon and the second cotyledon in the two-leaf-one-core stage of wheat, it was found that for cells with intact cell membranes, the amount of electrolyte transport across the membrane is linear within a specific time period. By detecting and calculating the changes in the conductivity of the exudate during this period through various parameters, the cell membrane permeability can be accurately reflected, and the repeatability and reliability of the results can meet the standards of quantitative research, that is, quantitative detection of cell membrane permeability is achieved; it is verified and summarized that the Δ conductivity method is used to detect cell membrane permeability, which is particularly suitable for qualitative and quantitative detection of plant cell membrane permeability. At the same time, the Δ conductivity method has application prospects in the quantitative detection of cell membrane permeability in large fungi and bacterial microorganisms. The present application provides a special conductivity detector to achieve automatic detection, avoid manual operation errors, and have high detection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of cell membrane permeability detection, and in particular to a cell membrane permeability Δ conductivity quantitative detection method and a conductivity detector. Background Art

[0002] Currently, cell membrane permeability is typically measured as relative permeability. Two primary testing methods exist: the vacuum method and the immersion method. Both methods provide relative permeability, qualitatively analyzing the permeability trend of a sample by comparing its relative permeability to a blank (CK) reference. However, in practical applications, existing testing methods often vary in their specific experimental procedures, making it difficult to guarantee consistent experimental conditions.

[0003] In the prior art, Xu Xinjuan et al. published a study in Jiangsu Agricultural Science, No. 7, 2014, comparing the immersion method and the air extraction method using leaves of five kinds of plants as materials. They proved that the air extraction time and the immersion time directly affected the relative conductivity values ​​of the leaves, and suggested that appropriate methods and times should be selected when testing different materials. Chen Aikui et al. published a study in Journal of Guangdong Education Institute, Vol. 30, No. 5, 2010, using wax gourd seedling leaves as materials. They found that the values ​​measured by the air extraction method were generally higher than those measured by the immersion method. They believed that the main reason for this was that the plant leaves were completely immersed in water after air extraction, and the electrolytes were more completely exuded. The relative conductivity values ​​of plants measured by the immersion method and the air extraction method showed the same trend of change. Yang Meng et al. published in Corn Science, 2012, 20(1): 90-94, that when measuring the relative conductivity of corn seedling leaves, the sample preparation, immersion time, cell killing method and time were all different. Although the relative conductivity values ​​could be used in their respective experiments, the experimental data of the two methods were not comparable due to different experimental conditions. Therefore, the currently commonly used cell membrane relative permeability test data is usually used for qualitative research, not quantitative research, and the consistency of repeated measurements cannot be well guaranteed. Therefore, developing a test method that can quantitatively detect cell membrane permeability and ensure the consistency of experimental conditions will greatly improve research efficiency and is of great significance. Summary of the Invention

[0004] In response to the technical problems in the prior art, the present invention aims to provide a method for quantitatively detecting cell membrane permeability using Δ conductivity. This method solves the problems of poor repeatability and inability to achieve quantitative detection of cell membrane permeability in the prior art, ensures consistency in experimental conditions, and establishes a database of various cell membrane permeabilities on this basis, greatly improving research efficiency. The method has good potential application prospects in the quantitative detection of plant cell membrane permeability and microbial cell membrane permeability. The present application also provides a conductivity detector specifically for the Δ conductivity method, which has high detection accuracy and significantly improved detection efficiency.

[0005] To achieve the above objectives, the technical solution provided by the present invention is:

[0006] In one aspect, the present invention provides a method for quantitatively detecting cell membrane permeability Δconductivity, comprising the following steps:

[0007] Step 1: Sample selection: Select plant samples as samples for cell membrane permeability testing;

[0008] Step 2: Conductivity data detection and collection; the sample selected in step 1 is cultured multiple times to obtain multiple test samples as biological replicates, multiple conductivity tests are performed on the same test sample as technical replicates, and multiple conductivity data of the multiple test samples are collected;

[0009] Step 3: Cell membrane permeability detection: Based on cells with intact cell membranes, the Δ conductivity method is used to qualitatively and quantitatively detect cell membrane permeability.

[0010] Based on the above technical solution, in the cell membrane permeability test in step 3, the cell membrane permeability unit is defined as: the conductivity value of the cell extravasation solution of 1g biomass with intact cell membrane in 1L pure water at a specific pressure for 1h, and the calculation formula is: Where, Δ conductivity value = σ 末 -σ 始 ; Solution volume refers to the volume of the solution tested, excluding the sample volume; Biomass = sample dry weight = sample wet weight × dry-to-wet weight ratio; Δ time = σ end time - σ start time.

[0011] Based on the above technical solution, the method for detecting cell membrane permeability includes the following steps:

[0012] S1, determine the detection time interval; continuous detection is based on the principle of a significant change in the measured conductivity, and the detection time interval is selected between 1 / 50 and 1 / 100 of the total time required for the first highest peak to appear;

[0013] S2, determine the time taken for Δ; select σ 始 and σ 末 Draw a scatter plot with time as the X axis and conductivity as the Y axis. Starting from the first point, select at least 6 consecutive points, add a linear trend line, and display R 2 If R 2 <0.99, then remove n points starting from the first point until R 2 ≥0.99, the number of retained points shall not be less than 6, and the first retained point shall be recorded as σ 始 Point, the last point is denoted as σ 末 point; from the beginning to detect σ 始 The total time of the point is recorded as σ 始 Time, from the beginning of detection to σ末 The total time of the point is recorded as σ 末 Time; Δ time = σ 末 Time-σ 始 Time

[0014] S3, determine biomass; select part of the sample as the sample for the Δ conductivity method cell membrane permeability test, dry the remaining sample to a constant weight, and calculate the dry-to-wet weight ratio; biomass = sample dry weight = sample wet weight × dry-to-wet weight ratio;

[0015] S4, determining the cell membrane integrity rate; using the trypan blue method or the FDA fluorescence staining method to detect the cell membrane integrity rate of the sample after the cell membrane permeability test, the cell membrane integrity rate is calculated.

[0016] Based on the above technical solution, when it is necessary to perform cell membrane permeability detection at a certain pressure value in step S1, it is necessary to determine the detection pressure value: the samples are processed in several pressure groups, and the appropriate test pressure and pressure reduction rate are selected based on the principle that the sample integrity rate is ≥50%. The sample integrity rate is detected using the trypan blue method or the FDA fluorescent staining method.

[0017] Based on the above technical solution, the Solarbio plant tissue staining kit was used in step S4 to detect the cell membrane integrity rate. The part of the leaf dyed blue was the damaged cell on the leaf. The calculation formula was:

[0018]

[0019] Based on the above technical solution, in step 1, the first cotyledon and the second cotyledon of wheat at the two-leaf-one-core stage are used as samples for cell membrane permeability detection, respectively recorded as sample A and sample B; the cultivation process is as follows:

[0020] Jimai 22 wheat seeds were selected, disinfected, and then soaked in ddH2O at 26°C for 24 hours, sown into seedling culture medium pots, and irrigated with Hoagland nutrient solution. They were then cultured in a constant temperature and light incubator under a cycle of 26°C for 16 hours of light and 16°C for 8 hours without light. Hoagland nutrient solution was replenished in a timely manner during the seedling raising period. When the seeds were grown to the two-leaf and one-cob stage, the first and second cotyledons were selected as samples A and B. After cleaning, the middle leaves of samples A and B were cut, respectively, and mixed thoroughly before use as samples.

[0021] Based on the above technical solution, the data collection in step 2 includes:

[0022] Taking three biological replicates and three technical replicates as an example, wheat seedlings were cultured three times as three biological replicates, 0.6g, 0.5g, and 0.4g fresh weight leaves were used as test samples, and the three biological replicates were marked as 0.6g group, 0.5g group, and 0.4g group respectively; then the conductivity values ​​of sample A and sample B were tested, and 3 samples were tested for each as 3 technical replicates; data collection was carried out after data detection was completed.

[0023] On the other hand, the present application also provides an application of a cell membrane permeability Δconductivity quantitative detection method in the quantitative detection of plant cell membrane permeability, as well as in the quantitative detection of cell membrane permeability in large fungi and bacterial microorganisms.

[0024] On the other hand, the present application also provides a conductivity detector for a quantitative detection method of cell membrane permeability Δ conductivity, characterized in that it includes a detection unit, a control unit and a pressure unit, the control unit is electrically connected to the detection unit and the pressure unit, and the pressure unit is connected to the detection unit through a pipeline to provide stable pressure for the detection unit.

[0025] Based on the above technical solution, the detection unit includes multiple pressure-resistant cavities, in which a control pump, a liquid-separating valve and a conductivity detector are arranged. The control pump is connected to the liquid-separating valve through a pipeline, and the head end of the liquid-separating valve is connected to the liquid storage container, and the end end is connected to the sample container in the conductivity detector.

[0026] The beneficial effects of the technical solution provided by the present invention are:

[0027] 1. The present invention studies the cell membrane permeability of the first and second cotyledons of wheat during the two-leaf, one-core stage and finds that for cells with intact cell membranes, the amount of electrolyte transport across the membrane is linear within a specific time period. By detecting and calculating the changes in the conductivity of the exudate during this period through various parameters, the cell membrane permeability can be accurately reflected. The repeatability and reliability of the results can meet the standards of quantitative research, that is, quantitative detection of cell membrane permeability can be achieved. Among them, indicating the cultivation conditions of the sample, the growth stage and sampling location during sampling, the appropriate pressure and pressure reduction rate during conductivity detection, etc. are necessary and critical to ensure the consistency of experimental conditions; the electrolytes carried by the sample, the test fluid, and the test container, and the electrolytes that overflow from the damaged cell membrane during the growth and production process, dissolve quickly in the test fluid, and the impact on the conductivity value can be effectively eliminated using the difference, i.e., Δ. Based on this research, the Δ conductivity method for detecting cell membrane permeability is verified and summarized, which is particularly suitable for qualitative and quantitative detection of plant cell membrane permeability.

[0028] 2. The present invention also provides a method for quantitatively detecting cell membrane permeability Δconductivity, which has potential application prospects in the quantitative detection of plant cell membrane permeability and the quantitative detection of cell membrane permeability in most microorganisms such as large fungi.

[0029] 3. The present invention also provides a conductivity detector dedicated to the quantitative detection method of cell membrane permeability Δconductivity, which can realize automatic sampling, sample mixing and automatic detection of multiple different samples. Compared with the manual operation used in the current detection methods, it avoids the errors caused by manual operation, and does not require multiple biological and technical repetitions to obtain accurate and reliable test results. At the same time, the detection time is reduced by at least 50%, and the detection efficiency is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a leaf scan of the first leaf of the 0.6g group sample after dyeing in the present invention;

[0031] Figure 2 This is a graph of the total area of ​​the first leaf of the 0.6g group sample in the present invention;

[0032] Figure 3 This is the staining area diagram of the first leaf of the 0.6g group sample in the present invention;

[0033] Figure 4 1 is a schematic diagram of the reliability statistical results of the Δ conductivity method, the air extraction method, and the immersion method in technical repetitions in the present invention; wherein, ** in the figure indicates that there is a very significant difference (P < 0.01) between the data groups in this method;

[0034] Figure 5 It is a structural schematic diagram of the conductivity detector in the present invention; DETAILED DESCRIPTION

[0035] The following will be combined with the contents of the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. The terms used in this specification are intended only to describe specific embodiments and are not intended to limit the present invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the listed items. It should be understood that, unless otherwise specified, the various raw materials in the present invention may be commercially available.

[0037] Example 1

[0038] In this embodiment, wheat is used as an example of a test sample to describe the specific detection process and principle of the Δ conductivity method.

[0039] This embodiment provides a method for quantitatively detecting cell membrane permeability Δconductivity, comprising the following steps:

[0040] Step 1: Sample selection: Select plant samples as samples for cell membrane permeability testing;

[0041] Step 2: Conductivity data detection and collection; the sample selected in step 1 is cultured multiple times to obtain multiple test samples as biological replicates, multiple conductivity tests are performed on the same test sample as technical replicates, and multiple conductivity data of the multiple test samples are collected;

[0042] Step 3: Cell membrane permeability detection: Based on cells with intact cell membranes, the Δ conductivity method is used to qualitatively and quantitatively detect cell membrane permeability.

[0043] Based on the above technical solution, in step 1, the first cotyledon and the second cotyledon of wheat at the two-leaf-one-core stage are used as samples for cell membrane permeability detection, respectively recorded as sample A and sample B; the cultivation process is as follows:

[0044] Jimai 22 wheat seeds were selected, disinfected, and then soaked in ddH2O at 26°C for 24 hours, sown into seedling culture medium pots, and irrigated with Hoagland nutrient solution. They were then cultured in a constant temperature and light incubator under a cycle of 26°C for 16 hours of light and 16°C for 8 hours without light. Hoagland nutrient solution was replenished in a timely manner during the seedling raising period. When the seeds were grown to the two-leaf and one-cob stage, the first and second cotyledons were selected as samples A and B. After cleaning, the middle leaves of samples A and B were cut, respectively, and mixed thoroughly before use as samples.

[0045] Preferably, during the sample cultivation, the seeds are disinfected by soaking in 75% ethanol for 20 minutes, and then the ethanol is rinsed off to complete the disinfection process; sample A and sample B are selected at the two-leaf and one-core stage of wheat 11 days after seedling cultivation; and leaves about 1 cm in the middle of the first cotyledon and the second cotyledon are selected.

[0046] In other more preferred embodiments, other plants such as tomatoes, corn, sea buckthorn, etc. can also be selected as detection objects. At the same time, it is not limited to the above-mentioned plant leaves, and the roots, stems and other parts of the plants can also be used as research objects. That is, the Δ conductivity method in this application for detecting cell membrane permeability has good universality and is widely used.

[0047] The present invention studies the cell membrane permeability of the first and second cotyledons of wheat during the two-leaf, one-core stage and finds that for cells with intact cell membranes, the amount of electrolyte transport across the membrane is linear within a specific time period. By detecting and calculating the changes in the conductivity of the exudate during this period through various parameters, the cell membrane permeability can be accurately reflected. The repeatability and reliability of the results can meet the standards of quantitative research, that is, quantitative detection of cell membrane permeability can be achieved. Among them, indicating the cultivation conditions of the sample, the growth stage and sampling site during sampling, the appropriate pressure and pressure reduction rate during conductivity detection, etc. are necessary and critical to ensure the consistency of experimental conditions; the electrolytes carried by the sample, the test fluid, and the test container, and the electrolytes that overflow from the damaged cell membrane during the growth and production process, dissolve quickly in the test fluid, and the impact on the conductivity value can be effectively eliminated using the difference, i.e., Δ. Based on this research, the cell membrane permeability Δ conductivity quantitative detection method, referred to as the Δ conductivity method, is verified and summarized to detect cell membrane permeability, which is particularly suitable for qualitative and quantitative detection of plant cell membrane permeability.

[0048] Based on the above technical solution, the data collection in step 2 includes:

[0049] Taking three biological replicates and three technical replicates as an example, wheat seedlings were cultured three times as three biological replicates, 0.6g, 0.5g, and 0.4g fresh weight leaves were used as test samples, and the three biological replicates were marked as 0.6g group, 0.5g group, and 0.4g group respectively; then the conductivity values ​​of sample A and sample B were tested, and 3 samples were tested for each as 3 technical replicates; data collection was carried out after data detection was completed.

[0050] In this example, three biological replicates and three technical replicates are used as examples for illustration. The conductivity values ​​are detected using a Ray-Magnetic DDSJ-318 conductivity meter, and data are collected using the Ray-Magnetic REXDCM data acquisition software V1.1.

[0051] It should be noted that the biological repetition mentioned above refers to the testing of the same indicator on wheat seedlings obtained from multiple cultures; technical repetition is multiple testing of the same sample for the same test indicator; biological repetition is conducive to eliminating the differences in test results caused by individual differences, and technical repetition is conducive to eliminating the errors in test results caused by differences in technical operations.

[0052] Based on the above technical solution, in the cell membrane permeability test in step 3, the cell membrane permeability unit is defined as: the conductivity value of the cell extravasation solution of 1g biomass with intact cell membrane in 1L pure water at a specific pressure for 1h, and the calculation formula is: Where, Δ conductivity value = σ 末 -σ 始; Solution volume refers to the volume of the solution tested, excluding the sample volume; Biomass = sample dry weight = sample wet weight × dry-to-wet weight ratio; Δ time = σ end time - σ start time.

[0053] This application uses the Δ conductivity method to define the cell membrane permeability unit, as described above. The biggest difference from the common vacuum method, immersion method, etc. in the prior art is that it only detects the cell membrane permeability of cells with intact cell membranes, and does not need to distinguish between dead and apoptotic cells. At the same time, it standardizes the experimental steps, value-taking methods, and permeability units, etc., to ensure the consistency of experimental conditions. In addition, by comparing two detection methods in the prior art, by detecting and comparing the cell membrane permeability of the first cotyledon and the second cotyledon in the two-leaf and one-core stage of wheat, the experimental results were statistically analyzed and found to be significantly superior to the existing detection methods in terms of repeatability. This proves its reliability in quantifying cell membrane permeability. The Δ conductivity method lays the foundation for the establishment of a cell membrane permeability database, and the establishment of a database will greatly improve research efficiency.

[0054] Preferably, the pressure value not marked with Q is detected at normal pressure by default; the unit of the cell membrane permeability is (μs / cm)·L / g·h; the unit of the Δ conductivity value is μs / cm; the unit of the solution volume is L; the unit of the biomass is g; and the unit of the Δ time is h.

[0055] Based on the above technical solution, the method for detecting cell membrane permeability includes the following steps:

[0056] S1, determine the detection time interval; continuous detection is based on the principle of a significant change in the measured conductivity, and the detection time interval is selected between 1 / 50 and 1 / 100 of the total time required for the first highest peak to appear;

[0057] S2, determine the time taken for Δ; select σ 始 and σ 末 Draw a scatter plot with time as the X axis and conductivity as the Y axis. Starting from the first point, select at least 6 consecutive points, add a linear trend line, and display R 2 If R 2 <0.99, then remove n points starting from the first point until R 2 ≥0.99, the number of retained points shall not be less than 6, and the first retained point shall be recorded as σ 始 Point, the last point is denoted as σ 末 point; from the beginning to detect σ 始 The total time of the point is recorded as σ 始 Time, from the beginning of detection to σ 末 The total time of the point is recorded as σ 末 Time; Δ time = σ 末 Time-σ 始 Time

[0058] S3, determine biomass; select part of the sample as the sample for the Δ conductivity method cell membrane permeability test, dry the remaining sample to a constant weight, and calculate the dry-to-wet weight ratio; biomass = sample dry weight = sample wet weight × dry-to-wet weight ratio;

[0059] S4, determining the cell membrane integrity rate; the cell membrane integrity rate of the sample after the cell membrane permeability test is tested using the trypan blue method or the FDA fluorescent staining method, and the cell membrane integrity rate is calculated. Based on the above technical solution, if the cell membrane permeability test in step S1 requires a certain pressure value, the test pressure value needs to be determined: the sample is processed in several pressure groups, and the appropriate test pressure and pressure reduction rate are selected based on the principle that the sample integrity rate is ≥50%. The sample integrity is tested using the trypan blue method or the FDA fluorescent staining method.

[0060] It is understood that other existing methods can also be used to detect the cell membrane integrity rate, as long as they can achieve accurate detection of the cell membrane integrity rate. In the preferred embodiment of the present application, the trypan blue method is used to detect the cell membrane integrity.

[0061] The above steps are not required when testing cell membrane permeability under normal pressure. If the test is not under normal pressure, it is necessary to determine the appropriate test pressure. The appropriate test pressure value should be confirmed through preliminary testing, that is, the samples should be processed in several pressure groups, and the appropriate test pressure should be selected based on the principle that the sample integrity rate is ≥50%.

[0062] Based on the above technical solution, the Solarbio plant tissue staining kit was used in step S4 to detect the cell membrane integrity rate. The part of the leaf dyed blue was the damaged cell on the leaf. The calculation formula was:

[0063]

[0064] Preferably, in this embodiment, the trypan blue method is used to detect the cell membrane integrity rate, that is, the leaves are stained using the Solarbio plant tissue staining kit, and the damaged cells on the sample leaves are counted according to the color development area mark to obtain the cell membrane integrity rate.

[0065] In this embodiment, the Δ conductivity method is used to detect cell membrane permeability, and the detection method is manually operated. The detection process is described using the 0.6g group as an example, as follows:

[0066] First, determine the pressure value and time interval in a preliminary test: preset the upper limit of the digital electric contact pressure gauge to 10 kPa and the lower limit to 60 kPa, where the start and stop of the vacuum pump is controlled by the pressure gauge; then prepare 6 vacuum tubes, add 100 ml of ddH2O to each tube for standby use; prepare 6 portions of 0.6 g of sample, add them to the sample tubes respectively, and place them in the vacuum tubes respectively.

[0067] Start the pressure gauge, and when the vacuum pressure reaches 60, 50, 40, 30, 20, and 10 kPa, close the valves of vacuum tubes 1 to 6 in sequence, and test the conductivity of each tube at intervals of 3-8 hours; repeat the start-up and testing until the first highest peak appears, take out each sample, and use the trypan blue method to test the cell membrane integrity rate; considering the test time and integrity rate, determine the test time interval to be 1-1.5 hours, and the test pressure value to be 30-40 kPa, that is, set the upper limit of the digital electric contact pressure gauge to 30 kPa and the lower limit to 40 kPa for standby use.

[0068] Secondly, collect the various parameters required for calculation in the Δ conductivity method: add 100ml of ddH2O to each vacuum tube for later use; prepare 0.6g groups of samples, and place 3 portions of sample A and sample B into 6 sample tubes respectively, and then place them into 6 vacuum tubes in turn. The vacuum tubes are sealed and set aside; after starting to test the conductivity, weigh the samples used to test the dry-to-wet weight ratio, record them, and then dry them to calculate the dry-to-wet weight ratio.

[0069] The conductivity test process is to start the digital electric contact pressure gauge, record the start time, and count down for 1 hour. After the first few vacuuming, there are many small bubbles overflowing from the sample, which need to be cleared in time; when the timing is up, close the vacuum tube ball valve, invert and mix the extravasated liquid in the vacuum tube, close the pressure gauge, open the air valve and immediately open the vacuum tube valve to let air in, balance the pressure to normal pressure, open the vacuum tube, and test the conductivity of the extravasated solution in tubes 1 to 6 in turn, and export the original data; repeat the test process. When σ ends, end the conductivity test and immediately test the cell integrity rate of sample A and sample B.

[0070] Furthermore, the cell membrane integrity rate was detected by using the Solarbio plant tissue staining kit, namely the trypan blue method. In this method, the blue-stained part of the leaf is the damaged cell on the leaf. After staining, the leaf was scanned into an image using an Epson Perfection V39Ⅱ scanner. Figure 1 As shown, the total leaf area is calculated as Figure 2 As shown, the stained area is Figure 3 As shown in the figure, according to the calculation formula of the completeness rate, Perform calculations.

[0071] Finally, the corresponding quantitative values ​​are calculated based on the parameters Δconductivity, Δtime, solution volume, biomass, and integrity rate obtained from the above tests or calculations, combined with the cell membrane permeability calculation formula provided above.

[0072] The above detection and calculation steps were repeated for the other two biological replicates, ie, the 0.4 g group and the 0.5 g group, using the Δ conductivity method to obtain the corresponding detection results of the cell membrane permeability of the other groups of samples.

[0073] The present invention also provides an application of a cell membrane permeability Δ conductivity quantitative detection method in the quantitative detection of plant cell membrane permeability, and in the quantitative detection of cell membrane permeability in large fungi and bacterial microorganisms.

[0074] Cell membrane permeability is an important physiological indicator for measuring plant resistance. It is widely used in research on plant drought resistance, cold resistance, salt and alkali resistance, disease resistance, food preservation, etc. It is also used in microbial research. However, because the diameter of large fungal spores, bacteria and other microorganisms is too small, special sample tubes are required for detection. According to the research on the quantitative detection of cell membrane permeability by the Δ conductivity method in this application, it is proved that in addition to the quantitative detection of plant cell membrane permeability, it also has potential application prospects in the quantitative detection of cell membrane permeability in most microorganisms such as large fungi and bacteria.

[0075] Example 2

[0076] On the basis of the above technical solution, the difference from the technical solution of embodiment 1 is that this embodiment uses a conductivity detector dedicated to the quantitative detection method of cell membrane permeability Δ conductivity, such as Figure 5 shown.

[0077] A conductivity detector for quantitative detection of cell membrane permeability Δconductivity includes a detection unit 1, a control unit 2, and a pressure unit 3. The control unit 2 is electrically connected to the detection unit 1 and the pressure unit 3. The pressure unit 3 is connected to the detection unit 1 through a pipeline to provide stable pressure for the detection unit 1.

[0078] Based on the above technical solution, the detection unit 1 includes multiple pressure-resistant cavities 11, in which a control pump 12, a liquid-separating valve 13 and a conductivity detector 14 are arranged. The control pump 12 is connected to the liquid-separating valve 13 through a pipeline. The head end of the liquid-separating valve 13 is connected to the liquid storage container, and the end end is connected to the sample container 141 in the conductivity detector 14.

[0079] The present application also provides a conductivity detector dedicated to the quantitative detection method of cell membrane permeability Δconductivity, which can realize automatic sampling, sample mixing and automatic detection of multiple different samples. Compared with the manual operation used in the current existing detection methods, it avoids the errors caused by manual operation, and does not require multiple biological repetitions and technical repetitions to obtain accurate and reliable test results. At the same time, the detection time is reduced by at least 50%, and the detection efficiency is greatly improved.

[0080] Specifically, the detection unit 1 is composed of multiple pressure-resistant cavities 11. A stable pressure can be maintained in each pressure-resistant cavity 11 through the pressure unit 3, and conductivity detection can be achieved under normal pressure, vacuum or pressurized conditions, providing a variety of detection environments to meet the detection requirements of different detection samples; it can be understood that each pressure-resistant cavity 11 is provided with a sealed door, and the outer wall of the cavity is provided with a hole for accommodating a valve and a connecting cable, which can be kept sealed.

[0081] A high-precision control pump 12 is provided in the pressure-resistant cavity 11 and is connected to a liquid separator valve 13. The liquid separator valve 13 is connected to the liquid in the liquid storage container, the detection liquid in the detector, and the waste liquid tank through pipelines. The control pump 12 controls which liquid enters and discharges the liquid separator valve 13; preferably, the control pump 12 can also mix the liquid in the sample container 141 in the conductivity detector 14 through pumping and releasing actions, without the need for manual mixing, and is more convenient to use. Preferably, the conductivity detector 14 is fixedly arranged in the pressure-resistant cavity and consists of three parts from top to bottom, which are detachably connected to each other and have good sealing after installation. The bottom is a sample container 141 and is connected to the liquid separation valve 13 through a pipeline to provide a test sample for the conductivity detection equipment in the middle; the middle is a conductivity detection equipment, which detects parameters such as conductivity and temperature of the sample. Preferably, the conductivity detection equipment can use the Leizhi DDSJ-318 conductivity meter to realize conductivity parameter detection; the top is a pressure balancing part, that is, a through hole is provided on the top pipeline for balancing pressure.

[0082] The control unit 2 is connected to a power source 4 and is electrically connected to the detection unit 1 and pressure unit 3 via cables. This controls the operation of various components of the conductivity tester, records various test parameters, and processes the test data according to the calculation formula of the Δ conductivity method described above. The pressure unit 3 is preferably a pressure pump, connected to the detection unit 1 via a pipeline, providing pressure and pressure regulation within the pressure-resistant chamber 11. The pressure pump is also electrically connected to the control unit via cables.

[0083] Comparative Example 1

[0084] As a comparative example, the present application also uses the vacuum method and the immersion method to qualitatively test the cell membrane permeability, as follows:

[0085] (1) Qualitative detection of cell membrane permeability using the vacuum method: prepare 6 clean and labeled 250ml Erlenmeyer flasks, add 100ml ddH2O, add the sample, and record the initial total weight; place in a vacuum desiccator, start the vacuum pump to evacuate for 10 minutes, slowly add air to normal pressure, take out the Erlenmeyer flask and observe whether all the leaves have sunk to the bottom; if not, repeat the vacuuming and degassing steps until all the leaves have sunk to the bottom; then let it stand at room temperature for 30 minutes, add ddH2O to the initial total weight, shake well and measure R1; heat in a boiling water bath for 30 minutes, cool to room temperature, add ddH2O to the initial total weight, shake well and measure R2; the calculation formula is: relative permeability of cell membrane = (R1 / R2) × 100%, where R1 represents the conductivity of the exudate before the leaves are killed; R2 represents the conductivity of the exudate after the leaves are killed.

[0086] (2) Qualitative detection of cell membrane permeability was performed using the immersion method: the sample was immersed in a full-temperature shaking incubator at 180 rpm and 26°C for 12 h, ddH2O was added to the initial total weight, and R1 was measured after shaking; the sample was heated in a boiling water bath for 30 min, cooled to room temperature, ddH2O was added to the initial total weight, and R2 was measured after shaking; the calculation formula was: relative permeability of cell membrane = (R1 / R2) × 100%, where R1 represents the conductivity of the exudate before the leaf is killed; R2 represents the conductivity of the exudate after the leaf is killed.

[0087] Experimental results and performance analysis

[0088] Due to differences in experimental data units, the data from the three assays are not comparable. Therefore, the formula A / B = (A permeability / B permeability) was used to calculate the A / B ratio, removing the units and making the three assays comparable. Three biological replicates were performed for each method using the same experimental conditions, yielding three data sets. These three data sets were derived from the ratios of the cell membrane permeability data for Sample A and Sample B (Group A: a1, a2, a3; Group B: b1, b2, b3). Each combined ratio contained nine values ​​(a1 / b1, a2 / b1, a3 / b1, a1 / b2, a2 / b2, a3 / b2, a1 / b3, a2 / b3, a3 / b3), totaling 27 values ​​for each method. A one-way ANOVA test was used to compare the reproducibility and reliability of the three assays at the biological and technical replicate levels. The specific test results are as follows.

[0089] (1) Repeatability of the Δ conductivity method

[0090] With name as factor and data as dependent variable, single factor ANOVA test was conducted on Δ conductivity method. The statistical results are shown in the table below; cell membrane permeability (Q (30KPa))A>B, which is related to the growth time of leaf cells. From the beginning of cell birth, the longer the growth time, the greater the chance of damage, and the closer the cell is to death, the greater the permeability of the cell membrane. There was no significant difference in the data between the groups (P>0.05), indicating that the Δ conductivity method only requires three technical replicates and no biological replicates are required.

[0091] Statistical table of single factor ANOVA test results using Δ conductivity method

[0092]

[0093] (2) Repeatability of the vacuum method and immersion method

[0094] Similarly, the name was used as the factor and the data was used as the dependent variable, and single-factor ANOVA tests were performed on the air extraction method A, B and the immersion method A, B. The statistical results are shown in the following table; it can be seen that the relative permeability of the cell membrane A>B, which is consistent with the test results of the Δ conductivity method, and also illustrates the reliability and accuracy of the Δ conductivity method proposed in this application. There was no significant difference in the data between the groups of air extraction method A (P>0.05), and there was a very significant difference in the data between the groups of air extraction method B, immersion method A, and immersion method B (P<0.01), indicating that only 3 technical repeats of air extraction method A are required without biological repeats, while biological repeats and technical repeats must be done for air extraction method B, immersion method A, and immersion method B. However, it is precisely because the air extraction method has the same significant problem of data difference as the immersion method when detecting B, so when using the air extraction method for detection, biological repeats and technical repeats are also required, just like the immersion method.

[0095] Statistical table of single factor ANOVA test results by vacuum method and immersion method

[0096]

[0097] (3) Reliability of the three methods at the biological replication level

[0098] A one-way ANOVA test was performed with the names of the three detection methods as factors and the corresponding data as dependent variables. The statistical results are shown in the table below. There was no significant difference in the final data between the Δ conductivity method and the vacuum method and the immersion method (p>0.05), indicating that the Δ conductivity method can achieve the same level of reliability as the existing detection methods, and also proves the reliability of the Δ conductivity method provided in this application.

[0099] Reliability statistical results of the Δ conductivity method, vacuum method, and immersion method in biological replicates

[0100]

[0101] (4) Comparison of the reproducibility of the three methods at the technical replication level

[0102] The biological replicate names of the three detection methods were used as factors and the corresponding data as dependent variables. Single-factor ANOVA tests were performed separately. The results are as follows: Figure 4 As shown; among them, there was no significant difference in the data between the groups using the Δ conductivity method (p>0.05), and there were very significant differences in the data between the groups using the vacuum method and the immersion method (p<0.01), indicating that the repeatability of the Δ conductivity method at the technical repetition level was significantly better than that of the vacuum method and the immersion method.

[0103] In summary, cell membrane permeability is an important physiological indicator for measuring plant resistance. It is widely used in research on plant drought resistance, cold resistance, salt and alkali resistance, disease resistance, food preservation, etc., and is also used in microbial research. The Δ conductivity method in this application does not need to distinguish between dead and apoptotic cells. It only detects cells with intact cell membranes, and indicates the sample culture conditions, the growth stage and the sampling site at the time of sampling to ensure the consistency of the experimental conditions, while ensuring the appropriate depressurization rate and pressure value. The Δ conductivity method can achieve the same level as the existing detection method in qualitative research, and the repeatability is significantly better than the existing detection method. At the same time, the Δ conductivity method in this application standardizes the experimental steps, value-taking methods and permeability units, etc., to ensure the consistency of experimental conditions and provide a basis for quantifying cell membrane permeability. Therefore, the Δ conductivity detection method for cell membrane permeability provided in this application can not only achieve excellent qualitative detection results, but also has obvious reliability in quantitative detection.

[0104] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from all perspectives, the embodiments should be regarded as illustrative and non-restrictive. The scope of the present invention is defined by the appended claims, not the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be included within the present invention. Any reference signs in the claims should not be construed as limiting the claim to which they relate.

[0105] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for quantitatively detecting cell membrane permeability Δ conductivity, characterized in that: The following steps are involved: Step 1: Sample selection: Select plant samples as samples for cell membrane permeability testing; Step 2: Conductivity data detection and collection; the sample selected in step 1 is cultured multiple times to obtain multiple test samples as biological replicates, multiple conductivity tests are performed on the same test sample as technical replicates, and multiple conductivity data of the multiple test samples are collected; Step 3: Cell membrane permeability test: Based on cells with intact cell membranes, the Δ conductivity method is used to qualitatively and quantitatively test the cell membrane permeability; the cell membrane permeability unit is defined as: the conductivity value of the cell extravasation solution of 1g biomass with intact cell membrane in 1L pure water at a specific pressure for 1h, and the calculation formula is: Q （压力值） = ; Among them, Δ conductivity value = σ 末 -σ 始 ; Solution volume is the volume of the solution tested, excluding the sample volume; Biomass = sample dry weight = sample wet weight × dry-wet weight ratio; Integrity rate = ;Δ time = σ end time - σ start time; The method for detecting cell membrane permeability comprises the following steps: S1, determine the detection time interval; continuous detection is based on the principle of a significant change in the measured conductivity, and the detection time interval is selected between 1 / 50 and 1 / 100 of the total time required for the first highest peak to appear; S2, determine the time taken for Δ; select σ 始 and σ 末 Draw a scatter plot with time as the X axis and conductivity as the Y axis. Starting from the first point, select at least 6 consecutive points, add a linear trend line, and display R 2 If R 2 <0.99, then remove n points starting from the first point until R 2 ≥0.99, the number of retained points shall not be less than 6, and the first retained point shall be recorded as σ 始 Point, the last point is denoted as σ 末 point; from the beginning to detect σ 始 The total time of the point is recorded as σ 始 Time, from the beginning of detection to σ 末 The total time of the point is recorded as σ 末 Time; Δ time = σ 末 Time-σ 始 Time S3, determine biomass; select part of the sample as the sample for the Δ conductivity method cell membrane permeability test, dry the remaining sample to a constant weight, and calculate the dry-wet weight ratio; biomass = sample dry weight = sample wet weight × dry-wet weight ratio; S4, determining the cell membrane integrity rate; using the trypan blue method or the FDA fluorescence staining method to detect the cell membrane integrity rate of the sample after the cell membrane permeability test, the cell membrane integrity rate is calculated.

2. A method for quantitatively detecting cell membrane permeability Δ conductivity according to claim 1, characterized in that: When cell membrane permeability testing needs to be performed at a certain pressure value in step S1, the test pressure value needs to be determined: the samples are processed in several pressure groups, and the appropriate test pressure and pressure reduction rate are selected based on the principle that the sample integrity rate is ≥50%. The sample integrity rate is detected using the trypan blue method or the FDA fluorescent staining method.

3. The method for quantitatively detecting cell membrane permeability Δ conductivity according to claim 1, wherein: In step S4, the Solarbio plant tissue staining kit is used to detect the cell membrane integrity rate. The parts of the leaves stained blue are damaged cells on the leaves. The calculation formula is: Completeness rate = .

4. The method for quantitatively detecting cell membrane permeability Δ conductivity according to claim 1, wherein: In step 1, the first cotyledon and the second cotyledon of wheat at the two-leaf-one-core stage are used as samples for cell membrane permeability detection, respectively recorded as sample A and sample B; the cultivation process is as follows: Jimai 22 wheat seeds were selected, disinfected, and then soaked in ddH2O at 26°C for 24 hours, sown into seedling culture medium pots, and irrigated with Hoagland nutrient solution. They were then cultured in a constant temperature and light incubator under a cycle of 26°C for 16 hours of light and 16°C for 8 hours without light. Hoagland nutrient solution was replenished in a timely manner during the seedling raising period. When the seeds were grown to the two-leaf and one-cob stage, the first and second cotyledons were selected as samples A and B. After cleaning, the middle leaves of samples A and B were cut, respectively, and mixed thoroughly before use as samples.

5. A method for quantitatively detecting cell membrane permeability Δ conductivity according to claim 4, characterized in that: The data collection in step 2 includes: Taking three biological replicates and three technical replicates as an example, wheat seedlings were cultured three times as three biological replicates, 0.6g, 0.5g, and 0.4g fresh weight leaves were used as test samples, and the three biological replicates were marked as 0.6g group, 0.5g group, and 0.4g group respectively; then the conductivity values ​​of sample A and sample B were tested, and 3 samples were tested for each as 3 technical replicates; data collection was carried out after data detection was completed.

6. Use of the method for quantitatively detecting cell membrane permeability Δconductivity according to any one of claims 1 to 5 in the quantitative detection of plant cell membrane permeability, and in the quantitative detection of cell membrane permeability in large fungi and bacterial microorganisms.

7. A conductivity detector for use in the method for quantitatively detecting cell membrane permeability Δ conductivity according to any one of claims 1 to 5, characterized in that: The conductivity detector comprises a detection unit, a control unit and a pressure unit. The control unit is electrically connected to the detection unit and the pressure unit to control the operation of various devices in the conductivity detector, record various detection parameters, and process the detection data according to the calculation formula of the Δ conductivity method. The pressure unit is connected to the detection unit through a pipeline to provide stable pressure for the detection unit.

8. The conductivity detector for quantitative detection of cell membrane permeability Δ conductivity according to claim 7, characterized in that: The detection unit includes multiple pressure-resistant cavities, each of which is equipped with a control pump, a liquid separation valve and a conductivity detector. The control pump is connected to the liquid separation valve through a pipeline. The head end of the liquid separation valve is connected to the liquid storage container, and the tail end is connected to the sample container in the conductivity detector.

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