CN117990661A - Bacterial activity detection method based on light scattering - Google Patents
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Abstract
The invention discloses a bacterial activity detection method based on light scattering, which is characterized in that a light intensity autocorrelation function is obtained by measuring the change of a light scattering signal, and then an exponential function fitting is carried out on the autocorrelation function to obtain an autocorrelation function, wherein the fitting of the autocorrelation function can obtain an attenuation rate, namely the attenuation speed of particle movement, and the attenuation rate reflects the movement speed of particles. The relationship between the attenuation rate and the bacterial activity of each bacterial suspension is established in advance, and then the information of the bacterial activity can be obtained by utilizing the attenuation rate obtained by measuring the light scattering device. This method does not require the cultivation of bacteria nor the use of special instruments and reagents. It can complete the measurement in a short time without damaging the bacteria, and thus can be used for monitoring the change of the activity of the bacterial solution for a long time.
Description
Technical Field
The invention belongs to the field of cell activity detection, and particularly relates to a bacterial activity detection method based on light scattering.
Background
Bacteria are an important group of microorganisms that have a complex impact on human health, both beneficial and detrimental. Most bacteria play a positive role in the daily life and physical health of humans, such as lactic acid bacteria and the like; however, some bacteria can cause a variety of diseases and infections, which pose a threat to human health. Therefore, bacterial activity detection has important application value in many fields, such as food industry, pharmaceutical industry, environmental monitoring, biomedical research, and the like.
The conventional bacterial activity detection method is a bacterial culture method, and generally comprises the steps of bacterial culture, cell counting and the like, wherein the method only can detect the number of living bacteria, the detection time is too long, and generally needs one day to several days, and many researches have shown that the culture method can only detect less than 1% of microorganisms in natural environment and can not detect bacteria and dead bacteria in a state of being "living but not being capable of being cultured" (VBNC). The current common bacterial activity detection methods comprise a fluorescent staining method, an enzyme-linked immunosorbent assay, a flow cytometry and the like, and compared with a culture method, the detection time is shortened by the methods, wherein living cells and dead cells are selectively marked by using fluorescent dyes by the fluorescent staining method, so that fluorescent signals with different colors are emitted by the living cells and the dead cells, the bacterial activity is detected, the proper fluorescent dyes are required to be selected according to bacterial types, a certain technical level and experimental equipment are required in the operation process, and the method depends on professionals, and in addition, the detection process is easy to cause damage to bacteria; the ELISA method utilizes the high selectivity of specific antibody to bacteria or metabolites thereof, can realize accurate detection of target bacteria, and needs to prepare specific antibody, and the detection process may have cross reaction with other substances, thus causing false positive results; flow cytometry allows high throughput analysis while measuring multiple parameters, but requires expensive flow cytometry equipment and technical support, requires special handling of samples, is highly demanding for samples, is sensitive to cell morphology and size, and may not be flexible enough for detection of certain specific morphologies of bacteria. In summary, these methods have high cost and complex operation required in the process of detecting bacterial activity, require a certain technical level and experimental equipment, rely on professionals, and in addition, the detection process is easy to cause damage to bacteria, so continuous monitoring cannot be realized.
Therefore, it is of great practical importance to develop a simple, rapid and non-destructive method for detecting bacterial activity. The detection method should be capable of measuring the activity of bacteria quickly and accurately without damaging the bacteria for long-term monitoring.
Disclosure of Invention
In order to overcome the defects in the prior art, the method for detecting the bacterial activity based on light scattering provides a simple, convenient, quick and nondestructive detection method. By finding the correlation between the light scattering signal and the bacterial activity, establishing a relational expression, and measuring the change of the light scattering signal, the information of the bacterial activity can be obtained. This method does not require the cultivation of bacteria nor the use of special instruments and reagents. It can complete the measurement in a short time without damaging the bacteria, and thus can be used for monitoring the change of the activity of the bacterial solution for a long time. The method overcomes the defects that the traditional culture method, the fluorescent staining method, the enzyme-linked immunosorbent assay method, the flow cytometry and other methods are complex in operation, time-consuming, possibly damaging bacteria, incapable of long-time measurement and the like.
The technical scheme of the invention realizes the optical detection of bacterial activity. The inventor mainly finds that some obvious motion differences exist between the living bacteria and the dead bacteria, such as living bacteria do rapid swimming in a solution, the dead bacteria do Brownian motion in the solution, the motion speeds are different, and the attenuation rates of the response in the scattered light autocorrelation function are different. Therefore, the characteristic can be utilized to construct the relationship between the attenuation rate and the bacterial activity, such as the proportional relationship between the attenuation rate and the bacterial activity, and the optical detection of the bacterial activity is realized.
The object of the invention is achieved by at least one of the following technical solutions.
A method for detecting bacterial activity based on light scattering, comprising the steps of:
1) Respectively measuring scattered light signals by a series of standard bacterial solutions with different bacterial activities and same total bacterial concentration, and obtaining a series of attenuation rates through signal autocorrelation treatment and data analysis;
2) Establishing a standard curve of attenuation rate and bacterial activity;
3) Operating the bacterial solution to be detected according to the step 1), measuring scattered light signals, obtaining attenuation rate through signal autocorrelation processing and data analysis, and calculating bacterial activity in the bacterial solution to be detected according to a standard curve.
Further, the scattered light signal is measured by a light scattering device; the light scattering device includes: an incident light source 1, a first lens 2, a sample bottle 3, a temperature-controlled sample stage 4, a second lens 5, an optical fiber 6, a photodetector 7 and a signal processor 8.
Further, the incident light source and the first lens are sequentially arranged on one side of the sample bottle, and the center of the incident light source and the center of the first lens are positioned on the same axis.
Further, the incident light source is converged by the first lens and vertically irradiates the sample bottle.
Furthermore, the temperature control sample stage comprises a base and a temperature control module, an inlet for placing a sample bottle is reserved at the top of the temperature control module, an incident light inlet and a scattered light outlet are reserved at two sides of the right angle respectively, and the rest part plays a role in controlling temperature and is used for providing the optimal survival temperature of bacteria.
Further, the centers of the optical fiber and the second lens are positioned on the same axis with the sample bottle and are perpendicular to the axis of the center of the incident light source and the center of the first lens.
Further, the light scattered by the sample is converged and injected into the optical fiber through the second lens, and the optical fiber is used for receiving the scattered light intensity.
Further, the photodetector is connected with the optical fiber, and is used for receiving the optical signal transmitted by the optical fiber and converting the optical signal into an electrical signal.
Further, the signal processor is used for receiving the electric signal converted by the photoelectric detector and fitting to obtain a light intensity autocorrelation function of scattered light, and calculating the attenuation rate according to the light intensity autocorrelation function; establishing a standard curve according to the attenuation rate of the standard bacterial solution in the step 1) and the bacterial activity of the standard bacterial solution known in the step (1); and (3) comparing the attenuation rate of the bacterial solution to be detected in the step (3) with the standard curve of the bacterial activity established in the step (2), and further outputting the activity information of the bacterial solution.
Further, the sample bottle is subjected to aseptic treatment for adding a sample; the aseptic processing mode of the sample bottle comprises ultraviolet sterilization or high-temperature high-pressure sterilization.
Furthermore, the sample bottle is arranged in the temperature control sample table, the temperature is adjusted to the optimal survival temperature of bacteria according to different bacteria types, and the measurement process is kept in a constant temperature state.
Further, the signal autocorrelation processing and data analysis comprise that a signal processor performs autocorrelation operation on an electric signal U (t) converted by a photoelectric detector to obtain a scattered light intensity autocorrelation function:
G(2)(τ)＝<U(t)U(t-τ)>，
Wherein t is the time of outputting the electric signal, tau is the delay time, U (t) is the electric signal output by the photoelectric converter at the time t, U (t-tau) is the electric signal output by the photoelectric converter at the time (t-tau), and the time is averaged; the variation of the scattered light intensity with time, called an optical signal, can be measured in the experiment, and the optical signal is converted into an electrical signal U (t) by a photodetector, both of which are available signals.
Normalizing the scattered light intensity autocorrelation function G (2) (tau) to obtain a normalized light intensity autocorrelation function:
Where Γ is the decay rate.
According to the formula, after the scattered light normalized light intensity autocorrelation function is obtained, the attenuation rate gamma is obtained by inversion operation, the attenuation rate gamma is related to bacterial motion in the solution, a larger attenuation rate corresponds to more live bacterial motion, a smaller attenuation rate corresponds to less live bacterial motion, a relation curve of the attenuation rate and activity is established in advance, and rapid measurement of bacterial activity can be achieved.
Further, the standard curve of the attenuation rate Γ and the bacterial activity V shows a linear relationship under the condition that the parameters of the light scattering device are fixed: Γ=k×v+b, where k is a scaling factor, and b is a base value, which is an average attenuation value obtained by measuring a dead bacterial solution of bacteria in a light scattering device in a plurality of measurements, depending on the type and size of the bacteria. Before measurement, the linear relation between the attenuation rate and the bacterial activity of the bacteria of the type to be measured is obtained through the light scattering device, and the linear relation is stored in the signal processor, and when the solution of the type to be measured is measured, the attenuation rate gamma is measured through the light scattering device, and then the bacterial activity is obtained through V=1/k (gamma-b) multiplied by 100%.
Compared with the prior art, the technical scheme of the invention has the advantages that:
according to the invention, the difference of the moving speeds of the living bacteria and the dead bacteria in the solution is utilized for the first time, so that the attenuation rate of the solution scattered light signal autocorrelation function is different, and the relationship between the living bacteria duty ratio and the attenuation rate can be constructed, so that the bacterial activity can be detected by an optical method. The method for detecting the bacterial activity by the optical method is simple to operate, low in cost and high in speed, and the bacteria cannot be damaged in the detection process, so that the method has obvious technical advantages compared with the existing culture method, fluorescent staining, enzyme-linked immunity and the like.
Compared with the prior art, the invention has the following beneficial effects:
(1) Quick and simple: the method is simple to operate, does not need to culture bacteria and complicated reagent preparation process, can finish measurement in a short time, and greatly saves time and manpower resources.
(2) Nondestructive testing: the laser intensity used by the light scattering device is far lower than the intensity which kills bacteria, so that bacteria cannot be damaged in the detection process, and the light scattering device can be used for monitoring the activity change of a bacterial solution for a long time.
(3) A variety of bacterial assays: the method can establish the relation between the activities and the attenuation rates of different bacteria, realizes the activity detection of various bacteria, and provides a more effective and convenient tool for scientific research and practical application.
(4) And (3) real-time monitoring: the method can be used for monitoring the activity change of the bacterial solution for a long time, so that the growth and death conditions of bacteria can be reflected in real time, and important reference basis is provided for scientific research, industrial production and medical practice.
In conclusion, the bacterial activity detection method based on light scattering provided by the invention has the advantages of rapidness, simplicity, no damage, multiple bacterial detection, real-time monitoring and the like, and provides a new solution for bacterial activity detection.
Drawings
FIG. 1 is a flow chart of the detection of bacterial activity, and A in FIG. 1 is a generalized flow chart of the detection; b in fig. 1 is a specific flowchart of each step.
Fig. 2 is a schematic view of a light scattering device.
FIG. 3 is a graph of normalized autocorrelation of scattered light intensity of E.coli of different activities.
FIG. 4 is a graph of E.coli activity Viability versus decay rate Γ.
FIG. 5 is a graph of normalized autocorrelation of scattered light intensity of different active Bacillus subtilis.
FIG. 6 is a graph of activity Viability of Bacillus subtilis versus the decay rate Γ standard.
Detailed Description
Specific implementations of the invention are further described below with reference to the drawings and examples, but the implementation and protection of the invention are not limited thereto. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used were not manufacturer-specific and were considered conventional products commercially available.
Fig. 2 is a schematic structural diagram of a light scattering device used in the present invention, wherein 1 is a laser, 2 is a first lens, 3 is a sample bottle, 4 is a temperature-controlled sample stage, 5 is a second lens, 6 is an optical fiber, 7 is a photodetector, and 8 is a signal processor.
In the following examples, the laser was selected from a He-Ne laser having a power of 5mW and a wavelength of 632.8nm, for generating an incident beam; the first lens is a convex lens and is positioned between the laser and the temperature control sample stage, and the focal point is positioned at the center of the temperature control sample stage by adjusting the placement position, so that the scattered light intensity is enhanced; the sample bottle is a round glass bottle with the light transmittance of more than 80%, and is placed in a temperature control sample table after high-temperature high-pressure sterilization; the temperature control sample stage is internally provided with a semiconductor refrigerating sheet with small volume and high efficiency, a control circuit and a radiating part thereof, and the temperature adjustment range is-10 ℃ to 70 ℃; the second lens is a convex lens and is arranged at a position where the included angle of scattered light of the sample solution is 90 degrees, and the scattered light is converged to the optical fiber and coupled to the photoelectric detector for detection; the optical fiber selects multimode optical fiber with good transmission characteristics; the signal processor comprises signal processing and data processing functions, the detected signals are subjected to autocorrelation processing and data analysis by the signal processor to obtain autocorrelation curve data of scattered light intensity, attenuation rate information is inverted and output, and the attenuation rate information is compared with an established standard curve of attenuation rate and bacterial activity to output bacterial activity information.
The basic principles of the signal autocorrelation process and data analysis are as follows:
the signal processor performs autocorrelation operation on the electric signal U (t) converted by the photoelectric detector to obtain a scattered light intensity autocorrelation function:
G(2)(τ)＝<U(t)U(t-τ)>，
Wherein, tau is delay time, U (t) is an electric signal output by the photoelectric converter at the moment t, U (t-tau) is an electric signal output by the photoelectric converter at the moment (t-tau), and the average value of time is represented; normalizing the scattered light intensity autocorrelation function G (2) (tau) to obtain a normalized light intensity autocorrelation function:
Wherein Γ is the decay rate;
Therefore, according to the method, the attenuation rate Γ can be obtained by obtaining the scattered light normalized light intensity autocorrelation function in an experiment, the attenuation rate Γ is related to bacterial motion in a solution, a larger attenuation rate corresponds to more live bacterial motion, a smaller attenuation rate corresponds to less live bacterial motion, a relation curve of the attenuation rate and activity is established in advance, and rapid measurement of bacterial activity can be achieved.
The standard curve of the attenuation rate gamma and the bacterial activity V shows a linear relation under the condition that the parameters of the light scattering device are certain: Γ=k×v+b, where k is a scaling factor, and b is a base value, which is an average attenuation value obtained by measuring a dead bacterial solution of bacteria in a light scattering device in a plurality of measurements, depending on the type and size of the bacteria. Before measurement, the linear relation between the attenuation rate and the bacterial activity of the bacteria of the type to be measured is obtained through the light scattering device, and the linear relation is stored in the signal processor, and when the solution of the type to be measured is measured, the attenuation rate gamma is measured through the light scattering device, and then the bacterial activity is obtained through V=1/k (gamma-b) multiplied by 100%.
Example 1
The detection flow chart of the bacterial activity detection is shown in fig. 1, wherein A in fig. 1 is a generalized detection flow chart; b in fig. 1 is a specific flowchart of each step. The detection method specifically comprises the following steps:
1) Calibration of a standard curve: inoculating Escherichia coli into nutrient broth and 70% isopropanol solution respectively, culturing for 12 hr, centrifuging to remove supernatant, washing the precipitate with physiological saline for 1-2 times to obtain live/dead bacteria solution; diluting the live/dead bacteria solution with physiological saline to a total bacteria concentration of 10 5 cells/ml;
2) Mixing the live bacteria solution and the dead bacteria solution according to different proportions to prepare a series of standard bacteria solutions with different activities (0%, 25%, 50%, 75%, 100%) and the same total concentration of bacteria;
3) Sequentially adding a series of standard bacterial solutions with different activities and same total concentration of bacteria into a sample bottle of the light scattering device;
4) Opening a laser to emit incident light into the sample bottle, converging scattered light results to an optical fiber by a second lens, and coupling the optical fiber to a photoelectric detector for detection;
5) The detected signal is subjected to autocorrelation processing and data analysis by a signal processor to obtain normalized autocorrelation curve data of scattered light intensity, and a series of attenuation rate information is outputted in an inversion mode;
6) Repeating the above experiment twice, linearly fitting the three groups of data, establishing a standard curve of the activity and the attenuation rate of the escherichia coli, and storing the standard curve in a signal processor.
7) Measurement of actual samples: and (3) taking the E.coli solution to be tested, operating according to the steps 3), 4) and 5), obtaining the attenuation rate, and comparing with the standard curve obtained in the step 6) to obtain the activity of the E.coli solution to be tested.
The normalized autocorrelation curves of the scattered light of the escherichia coli with different activity ratios, which are measured by the light scattering method, are shown in fig. 3, and it can be seen that the greater the escherichia coli activity is, the faster the autocorrelation curve is reduced along with time, namely, the greater the corresponding attenuation rate is.
The standard curve of the activity Viability-attenuation rate gamma of the escherichia coli measured by the light scattering method is shown in fig. 4, and the standard curve of the attenuation rate and the bacterial activity of the escherichia coli obtained by fitting three groups of experimental data is shown as follows:
Γ=0.3112v+96.3295, where the scaling factor k= 0.3112, the radix value b= 96.3295, the correlation index R 2 is 0.98073, the correlation is significant;
the comparison of the activity obtained by the E.coli solution to be tested obtained by the light scattering method with the actual activity is shown in Table 1, in which it can be seen that the light scattering method is suitable for measuring the bacterial activity. There is of course also some error, requiring subsequent fine adjustments to the measurement process or method, for example to improve the signal to noise ratio of the signal, etc.
TABLE 1
In the process of detecting the bacterial activity, the light scattering method only needs to establish a standard curve of the bacteria for the first time, the bacteria to be detected can be detected by simple concentration control, the measuring time is 3min, the operation difficulty and the time are far smaller than those of the traditional method, the laser intensity of the light scattering device is far lower than the intensity for killing the bacteria, the bacteria are not damaged in the detection process, and the method can be used for monitoring the change of the bacterial solution activity for a long time.
Example 2
The E.coli in example 1 was replaced with Bacillus subtilis; the temperature is changed to 30 ℃ suitable for the growth of bacillus subtilis, other operation steps are the same, and the experiment times are 1.
The normalized autocorrelation curves of the scattered light intensities of the bacillus subtilis with different activity ratios, which are detected by the light scattering method, are shown in a figure 5, and the figure shows that the greater the activity of the bacillus subtilis, the faster the autocorrelation curve is reduced along with time, namely the greater the corresponding attenuation rate is; the standard curve of the activity Viability-attenuation rate gamma of the bacillus subtilis is shown in fig. 6, and the standard curve of the attenuation rate and the bacterial activity of the bacillus subtilis is obtained by fitting experimental data in the graph, wherein the standard curve is as follows: Γ=0.2457v+81.0883, where the scaling factor k= 0.2457, the radix value b= 81.0883, the correlation index R2 is 0.98894, and the correlation is significant.
The above examples are only preferred embodiments of the present invention, and are merely for illustrating the present invention, not for limiting the present invention, and those skilled in the art should not be able to make any changes, substitutions, modifications and the like without departing from the spirit of the present invention.
Claims (10)
1. A method for detecting bacterial activity based on light scattering, comprising the steps of:
(1) Respectively measuring scattered light signals by a series of standard bacterial solutions with different bacterial activities and same total bacterial concentration, and obtaining a series of attenuation rates through signal autocorrelation treatment and data analysis;
(2) Establishing a standard curve of attenuation rate and bacterial activity;
(3) Operating the bacterial solution to be detected according to the step 1), measuring scattered light signals, obtaining attenuation rate through signal autocorrelation processing and data analysis, and calculating bacterial activity in the bacterial solution to be detected according to a standard curve.
2. The method for detecting bacterial activity based on light scattering according to claim 1, wherein the scattered light signal in step (1) is measured by a light scattering device; the light scattering device includes: the device comprises an incident light source, a first lens, a sample bottle, a temperature control sample stage, a second lens, an optical fiber, a photoelectric detector and a signal processor.
3. The method for detecting bacterial activity based on light scattering according to claim 2, wherein the incident light source and the first lens are sequentially arranged on one side of the sample bottle, and the center of the incident light source and the center of the first lens are positioned on the same axis; the incident light source is converged by the first lens and vertically irradiates into the sample bottle.
4. The method for detecting bacterial activity based on light scattering according to claim 2, wherein the temperature control sample stage comprises a base and a temperature control module, an inlet for placing a sample bottle is reserved at the top of the temperature control module, an incident light inlet and a scattered light outlet are reserved at two sides of the right angle respectively, and the rest part has a temperature control function and is used for providing an optimal bacterial survival temperature.
5. The method of claim 2, wherein the center of the optical fiber and the second lens are located on the same axis as the sample bottle and perpendicular to the axis of the center of the incident light source and the center of the first lens.
6. The method for detecting bacterial activity based on light scattering according to claim 2, wherein the light scattered by the sample is converged by the second lens and is incident on the optical fiber, and the optical fiber is used for receiving the scattered light intensity; the photoelectric detector is connected with the optical fiber and is used for receiving the optical signal transmitted by the optical fiber and converting the optical signal into an electric signal.
7. The method for detecting bacterial activity based on light scattering according to claim 2, wherein the signal processor is configured to receive the electrical signal converted by the photodetector and fit the electrical signal to obtain an intensity autocorrelation function of scattered light, and calculate the attenuation rate according to the intensity autocorrelation function; establishing a standard curve according to the attenuation rate of the standard bacterial solution in the step 1) and the bacterial activity of the standard bacterial solution known in the step (1); and (3) comparing the attenuation rate of the bacterial solution to be detected in the step (3) with the standard curve of the bacterial activity established in the step (2), and further outputting the activity information of the bacterial solution.
8. The method for detecting bacterial activity based on light scattering according to claim 2, wherein the sample bottle is subjected to a sterile treatment for adding a sample; the aseptic processing mode of the sample bottle comprises ultraviolet sterilization or high-temperature high-pressure sterilization; the sample bottle is arranged in the temperature control sample table, the temperature is regulated to the optimal survival temperature of bacteria according to different bacteria types, and the measurement process is kept in a constant temperature state.
9. The method for detecting bacterial activity based on light scattering according to claim 1, wherein the signal autocorrelation processing and data analysis include autocorrelation operation performed by a signal processor on an electrical signal U (t) converted by a photodetector to obtain an autocorrelation function of scattered light intensity:
G(2)(τ)＝<U(t)U(t-τ)>，
Wherein, the time is averaged, U (t) is the electric signal output by the photoelectric converter at the moment of t, U (t-tau) is the electric signal output by the photoelectric converter at the moment of (t-tau), t is the moment of outputting the electric signal, tau is the delay time, and G (2) (tau) is the scattered light intensity autocorrelation function; normalizing the scattered light intensity autocorrelation function G (2) (τ) to obtain a normalized light intensity autocorrelation function G (2) (τ):
Wherein Γ is the decay rate and τ is the delay time;
After the scattered light normalized light intensity autocorrelation function is obtained, the decay rate gamma is obtained by inversion operation, the decay rate gamma is related to bacterial motion in the solution, a larger decay rate corresponds to more live bacterial motion, a smaller decay rate corresponds to less live bacterial motion, a relation curve of the decay rate and activity is established in advance, and the rapid measurement of bacterial activity can be realized.
10. The method for detecting bacterial activity based on light scattering according to claim 9, wherein the decay rate Γ is linearly related to the bacterial activity V: Γ=k×v+b, where k is a scaling factor, and b is a base value, which is an average attenuation value obtained by measuring a dead bacterial solution of bacteria in a light scattering device in a plurality of measurements, depending on the type and size of the bacteria. Before measurement, the linear relation between the attenuation rate and the bacterial activity of the bacteria of the type to be measured is obtained through the light scattering device, the linear relation is stored in the signal processor, when the solution of the sample to be measured is measured, the attenuation rate gamma is measured through the light scattering device, and then the bacterial activity is obtained through V=1/k (gamma-b) multiplied by 100%.
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