CN112763558B - Staphylococcus aureus detection method - Google Patents
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Abstract
The invention discloses a staphylococcus aureus detection method. It comprises the following steps: s1: the method comprises the following steps of preparing staphylococcus aureus suspension liquid with different concentrations, detecting a characteristic value Y corresponding to the staphylococcus aureus suspension liquid with each concentration, and fitting the values to obtain a concentration calculation formula: y is 0.13 multiplied by LgX-0.01, and X is the concentration of the staphylococcus aureus suspension; s2: taking a staphylococcus aureus suspension to be detected, detecting a corresponding characteristic value Y of the staphylococcus aureus suspension, and calculating according to a concentration calculation formula: and (5) calculating the concentration of the staphylococcus aureus suspension to be detected when the Y is 0.13 multiplied by LgX-0.01. The method can accurately detect the concentration of the staphylococcus aureus and is simple to operate.
Description
Technical Field
The invention relates to the technical field of bacteria detection, in particular to a staphylococcus aureus detection method.
Background
Food-borne diseases refer to a general term for diseases which are caused by food contaminated by pathogenic bacteria entering human bodies by means of ingestion and causing human body infection or poisoning, and threaten human health and life safety. Although the development of modern science and technology reaches a certain level, food-borne diseases still seriously harm the health of people no matter in developed or developing countries, and the frequent food safety events in recent years also indicate that the food-borne diseases are not effectively controlled, so that the development of a novel detection technology for accurately detecting the food-borne pathogenic bacteria is very important, and the detection technology has important significance for preventing and controlling the food-borne diseases and guaranteeing the health of people.
Staphylococcus aureus can cause diseases such as neonatal meningitis, septicemia, necrotizing small intestine conjunctivitis and the like, and poses serious threats to human health and life safety, so that the research on the method for accurately detecting the Staphylococcus aureus has important significance. The existing staphylococcus aureus detection method generally adopts a chemical detection method, and has the defects of complex operation and poor repeatability
Disclosure of Invention
In order to solve the technical problems, the invention provides a staphylococcus aureus detection method which can accurately detect the concentration of staphylococcus aureus and is simple to operate.
In order to solve the problems, the invention adopts the following technical scheme:
the invention relates to a staphylococcus aureus detection method, which comprises the following steps:
s1: the method comprises the following steps of preparing staphylococcus aureus suspension liquid with different concentrations, detecting a characteristic value Y corresponding to the staphylococcus aureus suspension liquid with each concentration, and fitting the values to obtain a concentration calculation formula: y is 0.13 multiplied by LgX-0.01, and X is the concentration of the staphylococcus aureus suspension;
s2: taking a staphylococcus aureus suspension to be detected, detecting a corresponding characteristic value Y of the staphylococcus aureus suspension, and calculating according to a concentration calculation formula: and (5) calculating the concentration of the staphylococcus aureus suspension to be detected when the Y is 0.13 multiplied by LgX-0.01.
Preferably, the method for detecting the characteristic value Y corresponding to the staphylococcus aureus suspension at a certain concentration comprises the following steps:
m1: preparing a working electrode for cultivating staphylococcus aureus antigen, marking the working electrode which is not cultivated with the staphylococcus aureus antigen as a first working electrode, marking the working electrode cultivated with the staphylococcus aureus antigen as a second working electrode, forming a first electrochemical sensor array by the first working electrode, a counter electrode and a reference electrode, and forming a second electrochemical sensor array by the second working electrode, the counter electrode and the reference electrode;
m2: the same method is adopted to carry out m times of detection on the staphylococcus aureus suspension with the concentration, and the detection steps of each detection are as follows:
2.5 mul of golden yellow grape ball with the concentration is dripped on the first unused working electrode and the second unused working electrodeStanding the bacterial suspension for 25 minutes, respectively inserting the first electrochemical sensor array and the second electrochemical sensor array into two same buffer solutions, and sequentially switching n different scanning rates from small to large by adopting cyclic voltammetry to detect so as to obtain n reduction peak current difference values delta Ipw1、ΔIpw2…ΔIpwn，ΔIpwn＝Ip1wn-Ip2wn，ΔIpwnFor the reduction peak current difference at the nth scan rate in the w-th test, Ip1wnThe reduction peak current value corresponding to the first electrochemical sensor array at the nth scanning rate in the w detection is Ip2wnThe reduction peak current value corresponding to the second electrochemical sensor array under the nth scanning speed in the w detection is more than or equal to 1 and less than or equal to m;
m3: constructing a feature matrix D according to the detection data of m times of detection,
m4: performing quadratic spline interpolation on each line of data of the characteristic matrix D to obtain m curves x (t) corresponding to each line of data, performing the same data processing on the m curves x (t) to obtain m characteristic values F, wherein the data processing on each curve x (t) comprises the following steps:
bringing x (t) into a nonlinear oriented saturated resonance model
In (1),
function of saturation potential
The detected signal loading component of ing (t) coS (kt + η) + x (t),
wherein t is an interpolation variable, x (t) is a curve obtained by performing quadratic spline interpolation on a reduction peak current difference value obtained at a specific scanning speed, k is a real parameter, eta is a real parameter, nois (t) is colored noise with uneven power spectral density function, P is a real parameter, and Q is a real parameter,
drawing a resonance curve of the nonlinear directional saturated resonance model, taking the maximum peak value in the resonance curve as a resonance characteristic value E,
by the formula
Calculating to obtain a characteristic value F;
m5: and averaging the m characteristic values F to obtain an average value which is the value of the characteristic value Y.
In the process of detecting the staphylococcus aureus suspension for m times, a new unused first working electrode and a new unused second working electrode are adopted for each detection.
Preferably, the step M3 further includes the steps of:
calculating the stability factor lambdaij：
If λijIf the data is less than or equal to 0.8, judging that the data is abnormal, jumping to the step M2 to detect again,
if λijIf the data is more than 0.8, the data is judged to be normal, and the step M4 is executed.
Preferably, the n different scan rates comprise 50mV/s, 100mV/s, 200mV/s, 300mV/s, 400mV/s, 500 mV/s.
Preferably, the buffer solution is prepared by the following method: 0.5 mmol/1H2O2The solution is mixed with 1.0mol/1 Thi/HaC-NaAc solution according to the volume ratio of 1: 2.
Preferably, the first working electrode is a copper film electrode, and the second working electrode is prepared by the following method:
n1: mixing silver paste SL and carboxymethyl chitosan CMC (carboxymethyl chitosan) according to the mass ratio of 1: 3 to prepare 10ml of sol water solution, then carrying out ultrasonic dispersion for 15min, weighing 5mg of graphene oxide GO to dissolve in the sol water solution, carrying out ultrasonic oscillation for 25min to obtain GO/SL-CMC mixed solution, dripping 1.5 mu L of GO/SL-CMC mixed solution onto the surface of a copper film electrode, drying at room temperature for 5h, and forming a layer of GO/SL-CM film on the copper film electrode;
n2: soaking the copper membrane electrode in a solution of 1mmol/1 of thionine Thi for about 4min, assembling the thionine Thi on the surface of the copper membrane electrode, washing the copper membrane electrode with distilled water, washing off excessive thionine Thi, and standing and drying the copper membrane electrode;
n3: diluting the staphylococcus aureus polyclonal antibody solution by 300 times by using PBS buffer solution, then taking 4 mu l of the diluted staphylococcus aureus polyclonal antibody solution to be modified on a dried copper film electrode, and standing and drying the modified staphylococcus aureus polyclonal antibody solution in a drying dish;
n4: dripping 3.5 μ l of Staphylococcus aureus antigen solution on copper membrane electrode, culturing at 30 deg.C for 25min, and washing away unbound Staphylococcus aureus antigen on the copper membrane electrode with distilled water;
n5: mixing nanometer TiO2Mixing with water at a weight ratio of 0.1: 20, ultrasonically homogenizing, dripping 5 μ l of the mixture on a copper film electrode, and air drying to obtain a second working electrode.
Graphene oxide GO is selected as a working electrode modification material, carboxymethyl chitosan CMC and silver paste SL are used as dispersing agents to disperse the water-insoluble graphene oxide GO to enable the water-insoluble graphene oxide GO to be stably fixed on the surface of a working electrode, and then the water-insoluble graphene oxide GO is soaked in a biological dye thionine Thi to improve the electron transfer rate of the surface of the working electrode, and nano titanium dioxide TiO is adopted2Doping and irradiating with light to enhance response signal, fixing the staphylococcus aureus antibody on the modified working electrode, incubating staphylococcus aureus antigen on the prepared working electrode, and detecting the reduction peak current value by Cyclic Voltammetry (CV) under optimized detection conditions.
Preferably, in the step M2, during the detection by sequentially switching the n different scan rates from small to large by using cyclic voltammetry, visible light is irradiated onto the first working electrode and the second working electrode.
Preferably, the preparation method of the staphylococcus aureus antigen solution comprises the following steps: the concentration is 2 x 108cfu/ml～2×109The cfu/ml staphylococcus aureus is inactivated by 8 to 12 percent formalin at the temperature of between 25 and 39 ℃,and after inactivation, centrifuging to remove formalin, coating a flat plate for aseptic inspection, and after determining the sterility, resuspending the precipitate by using sterile normal saline with the same volume so as to obtain the staphylococcus aureus antigen solution.
Preferably, the preparation method of the staphylococcus aureus polyclonal antibody solution comprises the following steps:
after the rabbits are raised for 2 weeks, 15ml of blood is collected from the ear veins, and serum is taken out to be used as a negative serum sample; immunizing rabbit with Staphylococcus aureus antigen, performing second immunization at an interval of 3 days, enhancing immunity at an interval of 6 days, collecting blood from carotid artery at one time at 4 days after enhancing immunity, standing at room temperature for 45min, standing overnight at 4 deg.C, and centrifuging at 4 deg.C and 5000rpm for 45min the next day to obtain antiserum;
fixing 1.5ml affinity chromatography column on protein purifier, washing protective agent solution with deionized water, balancing column with PBS buffer solution, loading 1.5ml antiserum sample on column, eluting impurity with PBS buffer solution, and eluting staphylococcus aureus polyclonal antibody with citric acid buffer solution to obtain staphylococcus aureus polyclonal antibody solution.
The invention has the beneficial effects that: can accurately detect the concentration of staphylococcus aureus and has simple operation.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a schematic diagram of the resonance curve of the nonlinear directional saturation resonance model.
Detailed Description
The technical scheme of the invention is further specifically described by the following embodiments and the accompanying drawings.
Example (b): the staphylococcus aureus detection method of the embodiment, as shown in fig. 1, includes the following steps:
s1: the method comprises the following steps of preparing staphylococcus aureus suspension liquid with different concentrations, detecting a characteristic value Y corresponding to the staphylococcus aureus suspension liquid with each concentration, and fitting the values to obtain a concentration calculation formula: y is 0.13 multiplied by LgX-0.01, and X is the concentration of the staphylococcus aureus suspension;
s2: taking a staphylococcus aureus suspension to be detected, detecting a corresponding characteristic value Y of the staphylococcus aureus suspension, and calculating according to a concentration calculation formula: and (5) calculating the concentration of the staphylococcus aureus suspension to be detected when the Y is 0.13 multiplied by LgX-0.01.
In the scheme, a rectangular coordinate system is established by taking the concentration of the staphylococcus aureus suspension as an x axis and the characteristic value Y as a Y axis, corresponding points are marked in the rectangular coordinate system by the concentration of each staphylococcus aureus suspension and the corresponding characteristic value Y, and the points are fitted to obtain a concentration calculation formula: y is 0.13 × LgX-0.01.
The method for detecting the characteristic value Y corresponding to the staphylococcus aureus suspension at a certain concentration comprises the following steps:
m1: preparing a working electrode for cultivating staphylococcus aureus antigen, marking the working electrode which is not cultivated with the staphylococcus aureus antigen as a first working electrode, marking the working electrode cultivated with the staphylococcus aureus antigen as a second working electrode, forming a first electrochemical sensor array by the first working electrode, a counter electrode and a reference electrode, and forming a second electrochemical sensor array by the second working electrode, the counter electrode and the reference electrode;
m2: the same method is adopted to carry out m times of detection on the staphylococcus aureus suspension with the concentration, and the detection steps of each detection are as follows:
dripping 2.5 mul of staphylococcus aureus suspension with the concentration on a first unused working electrode and a second unused working electrode, standing for 25 minutes, respectively inserting a first electrochemical sensor array and a second electrochemical sensor array into two same buffer solutions, sequentially switching n different scanning rates from small to large by adopting a cyclic voltammetry method for detection, and irradiating visible light on the first working electrode and the second working electrode during detection to obtain n reduction peak current differences delta Ipw1、ΔIpw2…ΔIpwn，ΔIpwn＝Ip1wn-Ip2wn，ΔIpwnFor the difference in the reduction peak current at the nth scan rate in the w-th test, Ip1wnThe reduction peak current value corresponding to the first electrochemical sensor array at the nth scanning rate in the w detection is Ip2wnFor the w-th examinationMeasuring the reduction peak current value corresponding to the second electrochemical sensor array at the nth scanning speed, wherein w is more than or equal to 1 and less than or equal to m;
m3: constructing a feature matrix D according to the detection data of m times of detection,
calculating the stability factor lambdaij：
If λijIf the data is less than or equal to 0.8, judging that the data is abnormal, jumping to the step M2 to detect again,
if λijIf the data is more than 0.8, judging that the data is normal, and executing a step M4;
m4: performing quadratic spline interpolation on each line of data of the characteristic matrix D to obtain m curves x (t) corresponding to each line of data, performing the same data processing on the m curves x (t) to obtain m characteristic values F, wherein the data processing on each curve x (t) comprises the following steps:
bringing x (t) into a nonlinear oriented saturated resonance model
In (1),
function of saturation potential
The detected signal loading component of ing (t) coS (kt + η) + x (t),
wherein t is an interpolation variable, x (t) is a curve obtained by performing quadratic spline interpolation on a reduction peak current difference value obtained at a specific scanning speed, k is a real parameter, eta is a real parameter, nois (t) is colored noise with uneven power spectral density function, P is a real parameter, and Q is a real parameter,
as shown in fig. 2, a resonance curve of the nonlinear directional saturated resonance model is plotted, the maximum peak value in the resonance curve is taken as the resonance characteristic value E,
by the formula
Calculating to obtain a characteristic value F;
m5: and averaging the m characteristic values F to obtain an average value which is the value of the characteristic value Y.
In the process of detecting the staphylococcus aureus suspension for m times, a new unused first working electrode and a new unused second working electrode are adopted for each detection.
The n different scan rates include 50mV/s, 100mV/s, 200mV/s, 300mV/s, 400mV/s, and 500 mV/s.
The preparation method of the buffer solution comprises the following steps: 0.5mmol/l of H2O2The solution is mixed evenly with 1.0mol/l Thi/HaC-NaAc solution according to the volume ratio of 1: 2.
The first working electrode is a copper film electrode, the counter electrode is a Pt electrode, and the reference electrode is an Ag/AgCl electrode.
The second working electrode was prepared as follows:
n1: mixing silver paste SL and carboxymethyl chitosan CMC according to the mass ratio of 1: 3 to prepare 10ml of sol aqueous solution, then carrying out ultrasonic dispersion for 15min, weighing 5mg of graphene oxide GO to dissolve in the sol aqueous solution, carrying out ultrasonic oscillation for 25min to obtain GO/SL-CMC mixed solution, dripping 1.5 mu L of GO/SL-CMC mixed solution onto the surface of a copper film electrode, drying at room temperature for 5h, and forming a layer of GO/SL-CM film on the copper film electrode;
n2: soaking the copper membrane electrode in a 1mmol/1 thionine Thi solution for about 4min, assembling the thionine Thi on the surface of the copper membrane electrode, washing the copper membrane electrode with distilled water, washing off redundant thionine Thi, and standing and drying the copper membrane electrode;
n3: diluting the staphylococcus aureus polyclonal antibody solution by 300 times by using PBS buffer solution, then taking 4 mu l of the diluted staphylococcus aureus polyclonal antibody solution to be modified on a dried copper film electrode, and standing and drying the modified staphylococcus aureus polyclonal antibody solution in a drying dish;
n4: dripping 3.5 μ l of Staphylococcus aureus antigen solution on copper membrane electrode, culturing at 30 deg.C for 25min, and washing away unbound Staphylococcus aureus antigen on the copper membrane electrode with distilled water;
n5: mixing nanometer TiO2Mixing with water at a weight ratio of 0.1: 20, ultrasonically homogenizing, dripping 5 μ l of the mixture on a copper film electrode, and air drying to obtain a second working electrode.
Graphene oxide GO is selected as a working electrode modification material, carboxymethyl chitosan CMC and silver paste SL are used as dispersing agents to disperse the water-insoluble graphene oxide GO to enable the water-insoluble graphene oxide GO to be stably fixed on the surface of a working electrode, and then the water-insoluble graphene oxide GO is soaked in a biological dye thionine Thi to improve the electron transfer rate of the surface of the working electrode, and nano titanium dioxide TiO is adopted2Doping and irradiating with light to enhance response signal, fixing the staphylococcus aureus antibody on the modified working electrode, incubating staphylococcus aureus antigen on the prepared working electrode, and detecting the reduction peak current value by Cyclic Voltammetry (CV) under optimized detection conditions.
The preparation method of the staphylococcus aureus antigen solution comprises the following steps: the concentration is 2 x 108cfu/ml～2×109Inactivating cfu/ml staphylococcus aureus by 8-12% formalin at 25-39 ℃, centrifuging to remove the formalin after inactivation, coating a flat plate for aseptic inspection, and resuspending a precipitate by using isometric sterile normal saline after determining the sterility so as to obtain a staphylococcus aureus antigen solution.
The preparation method of the staphylococcus aureus polyclonal antibody solution comprises the following steps:
after the rabbits are raised for 2 weeks, 15ml of blood is collected from the ear veins, and serum is taken out to be used as a negative serum sample; immunizing rabbit with Staphylococcus aureus antigen, performing second immunization at an interval of 3 days, enhancing immunity at an interval of 6 days, collecting blood from carotid artery at one time at 4 days after enhancing immunity, standing at room temperature for 45min, standing overnight at 4 deg.C, and centrifuging at 4 deg.C and 5000rpm for 45min the next day to obtain antiserum;
fixing 1.5ml affinity chromatography column on protein purifier, washing protective agent solution with deionized water, balancing column with PBS buffer solution, loading 1.5ml antiserum sample on column, eluting impurity with PBS buffer solution, and eluting staphylococcus aureus polyclonal antibody with citric acid buffer solution to obtain staphylococcus aureus polyclonal antibody solution.
Claims (5)
1. A staphylococcus aureus detection method is characterized by comprising the following steps:
s1: the method comprises the following steps of preparing staphylococcus aureus suspension liquid with different concentrations, detecting a characteristic value Y corresponding to the staphylococcus aureus suspension liquid with each concentration, and fitting the values to obtain a concentration calculation formula: y is 0.13 multiplied by LgX-0.01, and X is the concentration of the staphylococcus aureus suspension;
s2: taking a staphylococcus aureus suspension to be detected, detecting a corresponding characteristic value Y of the staphylococcus aureus suspension, and calculating according to a concentration calculation formula: y is 0.13 multiplied by LgX-0.01, and the concentration of the staphylococcus aureus suspension to be detected is calculated;
the method for detecting the characteristic value Y corresponding to the staphylococcus aureus suspension at a certain concentration comprises the following steps:
m1: preparing a working electrode for cultivating staphylococcus aureus antigen, marking the working electrode which is not cultivated with the staphylococcus aureus antigen as a first working electrode, marking the working electrode cultivated with the staphylococcus aureus antigen as a second working electrode, forming a first electrochemical sensor array by the first working electrode, a counter electrode and a reference electrode, and forming a second electrochemical sensor array by the second working electrode, the counter electrode and the reference electrode;
m2: the same method is adopted to carry out m times of detection on the staphylococcus aureus suspension with the concentration, and the detection steps of each detection are as follows:
dripping 2.5ul of staphylococcus aureus suspension with the concentration on the unused first working electrode and the unused second working electrode, standing for 25 minutes, respectively inserting the first electrochemical sensor array and the second electrochemical sensor array into two same buffer solutions, and sequentially switching n different scanning rates from small to large by adopting a cyclic voltammetry method for detection to obtain n reduction peak current differences delta Ipw1、ΔIpw2…ΔIpwn，ΔIpwn＝Ip1wn-Ip2wn，ΔIpwnFor the reduction peak current difference at the nth scan rate in the w-th test, Ip1wnThe reduction peak current value corresponding to the first electrochemical sensor array at the nth scanning rate in the w detection is Ip2wnThe reduction peak current value corresponding to the second electrochemical sensor array under the nth scanning speed in the w detection is more than or equal to 1 and less than or equal to m;
m3: constructing a feature matrix D according to the detection data of m times of detection,
m4: performing secondary spline interpolation on each row of data of the feature matrix D to obtain m curves x (t) corresponding to each row of data, performing the same data processing on the m curves x (t) to obtain m feature values F, wherein the data processing on each curve x (t) comprises the following steps: bringing x (t) into a nonlinear oriented saturated resonance model
Medium and saturated potential function
The detection signal loading component of (t) cos (kt + η) + x (t),
wherein t is an interpolation variable, k is a real parameter, η is a real parameter, nois (t) is colored noise having a power spectral density function that is not flat, P is a real parameter, Q is a real parameter,
drawing a resonance curve of the nonlinear directional saturated resonance model, taking the maximum peak value in the resonance curve as a resonance characteristic value E,
by the formula
Calculating to obtain a characteristic value F;
m5: and averaging the m characteristic values F to obtain an average value which is the value of the characteristic value Y.
2. The staphylococcus aureus detection method of claim 1, wherein the step M3 further comprises the steps of:
calculating the stability factor lambdaij：
If λijIf the data is less than or equal to 0.8, judging that the data is abnormal, jumping to the step M2 to detect again,
if λijIf the data is more than 0.8, the data is judged to be normal, and the step M4 is executed.
3. The method according to claim 1, wherein the n different scan rates comprise 50mV/s, 100mV/s, 200mV/s, 300mV/s, 400mV/s, and 500 mV/s.
4. The method for detecting staphylococcus aureus according to claim 1, wherein the buffer solution is prepared by the following steps: 0.5 mmol/1H2O2The solution is mixed evenly with 1.0mol/l Thi/HaC-NaAc solution according to the volume ratio of 1: 2.
5. The method for detecting staphylococcus aureus according to claim 1, wherein the working electrode is a copper membrane electrode, the counter electrode is a Pt electrode, and the reference electrode is an Ag/AgC1 electrode.
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