CN111398388B - Method for detecting Listeria monocytogenes based on bifunctional sensor for detecting nucleic acid hly and acetoin - Google Patents
Method for detecting Listeria monocytogenes based on bifunctional sensor for detecting nucleic acid hly and acetoin Download PDFInfo
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Abstract
The invention discloses a method for detecting listeria monocytogenes based on a bifunctional sensor for detecting nucleic acid hly and metabolic marker acetoin, which comprises the steps of (1) preparing the bifunctional sensor for detecting listeria monocytogenes nucleic acid and metabolic marker; (2) calibrating or calibrating the dual-function sensor; (3) taking a milk sample as an example, the method detects the concentration of listeria monocytogenes in milk by using a standard-adding recovery method based on a dual-function sensor. The method for detecting different markers is integrated in a single platform, simultaneous detection and mutual authentication of different markers of the listeria monocytogenes are realized by using one dual-function sensor, the detection accuracy is enhanced, and meanwhile, the method shortens the identification time of the listeria monocytogenes of about 2-5 days in the national standard GB4789.30-2016 to about 1.5 hours, saves time and power, and has wide application prospect.
Description
Technical Field
The invention relates to a method for detecting Listeria monocytogenes, in particular to a method for detecting Listeria monocytogenes based on a bifunctional sensor for detecting nucleic acid hly and metabolic marker acetoin. Belongs to the field of electrochemical analysis and test.
Background
Listeria monocytogenes (Listeria monocytogenes), abbreviated Listeria monocytogenes, is a gram-positive bacterium which was first described in 1926 in a disease affecting rabbits and guinea pigs. It was considered a causative factor of human disease in the 70's of the 20 th century and was subsequently identified as a food-borne pathogen in the 80's. Listeriosis is a disease caused by the ingestion of food contaminated with listeriosis monocytogenes, mainly affecting immunocompromised patients, pregnant women and newborns. After ingestion of contaminated food, listeria monocytogenes crosses the intestinal, blood-brain and placental barriers, causing gastroenteritis, meningitis, septicemia, abortion, pregnancy complications, etc. In addition, the listeria monocytogenes can tolerate more extreme environments, such as a wider temperature range (1-45 ℃), a wider pH range (4.5-9.6), a high salt concentration (10% -15%), and the like, and the characteristics enable the listeria monocytogenes to coexist with different pathogenic microorganisms in different types of foods, thereby seriously harming human health. Therefore, the reliable, sensitive and accurate detection of the listeria monocytogenes in the food has important significance for preventing infection.
Extensive research in recent decades has provided a variety of methods for detecting listeria monocytogenes. Among them, the method based on isolated culture is known as "gold standard" for detecting Listeria monocytogenes. However, the method requires a certain professional skill of a researcher, and the identification steps are time-consuming (2-5 days), tedious (including kinetic experiments, catalase tests, sugar fermentation, hemolytic experiments and the like). In view of the disadvantages of the isolation culture detection method, researchers have developed many more rapid detection methods, such as enzyme-linked immunosorbent assay (ELISA), DNA hybridization, and real-time fluorescent quantitative polymerase chain reaction (qPCR). Although these methods based on detection of a single marker (antigen or marker gene) of listeria monocytogenes can rapidly detect listeria monocytogenes, there is also a probability of false positive or false negative. For this reason, the inspector usually needs to repeat a single detection method for many times, or different detection methods are used in combination to enhance the accuracy of the detection of listeria monocytogenes. However, as the detection accuracy is improved, a number of problems are generated, such as a long identification period, various expensive instruments and equipment required for different methods, and limited use of non-laboratory scenes. Based on the method, establishing an accurate, simple, specific and rapid analysis means to detect the listeria monocytogenes in the food has important practical significance and market demand.
Disclosure of Invention
Aiming at the defects of the existing method for detecting the listeria monocytogenes, the invention aims to provide a method for detecting the listeria monocytogenes based on a bifunctional sensor for detecting a nucleic acid marker hly and a metabolic marker acetoin of the listeria monocytogenes.
The invention discloses a method for detecting Listeria monocytogenes based on a bifunctional sensor for detecting nucleic acid hly and acetoin, which comprises the following steps:
(1) preparing a bifunctional sensor based on detection of listeria monocytogenes nucleic acid and metabolic markers;
(2) calibrating or calibrating the dual-function sensor;
(3) taking a milk sample as an example, detecting the concentration of listeria monocytogenes in milk by using a standard-adding recovery method based on a dual-function sensor;
the method is characterized in that:
the preparation method of the bifunctional sensor based on detection of the listeria monocytogenes nucleic acid and the metabolic marker in the step (1) comprises the following steps:
1) preparing a nanoporous gold (NPG)/Glassy Carbon Electrode (GCE): placing an Au/Ag alloy sheet with the thickness of 100 +/-10 nm in concentrated nitric acid, corroding for 1 hour at the temperature of 40 +/-2 ℃ to prepare a nano porous gold film, fixing the prepared nano porous gold film on the surface of a glassy carbon electrode, and drying for 1-2 hours in vacuum to prepare an NPG/GCE electrode;
2) preparation of ssDNA/NPG/GCE electrodes: dissolving ssDNA (single-stranded deoxyribonucleic acid) of a capture probe which is partially complementary with an hly gene and is modified with sulfydryl at the tail end into ultrapure water to prepare a ssDNA solution with the concentration of 0.5-3 mu M, then immersing the NPG/GCE electrode into the ssDNA solution, and incubating for 48-72 hours at 4 ℃ to prepare the ssDNA/NPG/GCE electrode; wherein the sequence of the capture probe ssDNA is 5' -TGGCGGCACATTTGTCACTGCA- (CH2)3-SH-3’；
3) Preparing a BSA/ssDNA/NPG/GCE electrode of the bifunctional sensor: incubating the prepared ssDNA/NPG/GCE electrode and Bovine Serum Albumin (BSA) with the concentration of 0.5-5% m/v at 4-30 ℃ for 15-30 minutes to prepare a difunctional sensor BSA/ssDNA/NPG/GCE electrode based on detection of Listeria monocytogenes nucleic acid and metabolic markers;
the method for calibrating or calibrating the dual-function sensor in the step (2) comprises the following steps:
1) detection of nucleic acid marker hly: the cultured concentration is 104～109CFU mL-1The listeria monocytogenes solution ofTaking 1-10 mL of the solution with the same concentration, ultrasonically breaking cells and long-chain nucleic acid at the frequency of 20-60 kilohertz, and incubating the cells and the long-chain nucleic acid with a BSA/ssDNA/NPG/GCE electrode at the temperature of 20-30 ℃ for 5-30 minutes to prepare a hly/BSA/ssDNA/NPG/GCE electrode; incubating hly/BSA/ssDNA/NPG/GCE electrode with 5-40 mu M Methylene Blue (MB) at 20-30 ℃ for 5-30 minutes to prepare an MB/hly/BSA/ssDNA/NPG/GCE electrode; putting an MB/hly/BSA/ssDNA/NPG/GCE electrode serving as a working electrode, a platinum electrode serving as a counter electrode and a saturated calomel electrode serving as a reference electrode into a reaction system of 50mM phosphate buffer solution, and performing linear sweep voltammetry by using 20-100 mV s-1Detecting the scanning speed in a voltage range of-0.5 to +0.1V, and drawing the obtained MB peak current density value to the concentration of the Listeria monocytogenes to obtain a linear standard curve;
2) detection of metabolic marker acetoin: firstly, establishing a linear relation of acetoin concentration to NADH reduction amount, adding the acetoin into a 50mM phosphate buffer solution reaction system containing 1-5 mM NADH according to different concentrations of 0-500 mu M, taking a BSA/ssDNA/NPG/GCE electrode as a working electrode, a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode, and utilizing a linear sweep voltammetry method to obtain a sample with the concentration of 20-100 mV s-1The sweep rate of (d) was measured in the range of +0.1 to +0.7V, and the obtained NADH initial peak current value was used as a control (j)NADH control) (ii) a Then adding 1-5 mg of heterologously expressed acetoin reductase into a buffer system, reacting for 5-30 minutes at 20-30 ℃, then taking a BSA/ssDNA/NPG/GCE electrode as a working electrode, and utilizing a linear scanning voltammetry to obtain a sample with the concentration of 20-100 mV s-1The sweep rate is detected within a voltage range of +0.1 to +0.7V, and the obtained peak current value of NADH after the reaction is taken as jNADH reactionWill j isNADH controlMinus jNADH reactionObtaining delta j, and drawing the delta j obtained by detecting acetoin with different concentrations to the concentration of the acetoin to obtain a linear standard curve of the concentration of the acetoin; then establishing a linear relation of acetoin concentration to Listeria monocytogenes concentration, and taking 1-50 mL of cultured solution with the concentration of 8.0 multiplied by 105～3.5×109CFU mL-1The listeria monocytogenes liquid is prepared by centrifuging bacterium liquids with different concentrations at 4000-8000 rpm, collecting culture medium supernatant, adding 50mM phosphate buffer solution reaction system, and adding into the culture medium supernatantAdding NADH with the final concentration of 1-5 mM into the system, detecting the concentration of the acetoin contained in the supernatant of the Listeria monocytogenes with different concentrations according to a method for establishing a linear relation of the concentration of the acetoin to the reduction amount of the NADH, and drawing a linear standard curve of the concentration of the acetoin to the concentration of the Listeria monocytogenes;
taking the milk sample as an example in the step (3), the method for detecting the concentration of listeria monocytogenes in milk by using a standard-adding recovery method based on the dual-function sensor comprises the following steps:
adding 25mL of finished milk into 225mL of culture medium suitable for growth of Listeria monocytogenes, and naming the solution as solution 1; 0.1mL of the solution was removed from solution 1 and added to 10mL of a medium suitable for growth of Listeria monocytogenes, designated solution 2; adding appropriate amount of Listeria monocytogenes into the solution 2 to make the final concentration of the bacteria be 104CFU mL-1Culturing at 37 ℃ for 6 hours, 9 hours and 12 hours, detecting the nucleic acid marker and the metabolic marker of the listeria monocytogenes by using the BSA/ssDNA/NPG/GCE electrode prepared in the step (1) according to the method in the step (2), and respectively substituting the obtained peak current values into different linear standard curves to correspondingly obtain the concentration of the listeria monocytogenes.
In the method for detecting listeria monocytogenes by using the bifunctional sensor for detecting nucleic acid hly and acetoin, the method for preparing the bifunctional sensor for detecting listeria monocytogenes nucleic acid and metabolic marker in step (1) preferably comprises:
1) preparing a nanoporous gold (NPG)/Glassy Carbon Electrode (GCE): placing an Au/Ag alloy sheet with the thickness of 100nm in concentrated nitric acid, corroding for 1 hour at the temperature of 40 ℃ to prepare a nano porous gold film, fixing the prepared nano porous gold film on the surface of a glassy carbon electrode, and drying for 1 hour in vacuum to prepare an NPG/GCE electrode;
2) preparation of ssDNA/NPG/GCE electrodes: dissolving ssDNA (single stranded deoxyribonucleic acid) of a capture probe which is partially complementary with an hly gene and is modified with sulfydryl at the tail end in ultrapure water to prepare a ssDNA solution with the concentration of 1 mu M, then immersing an NPG/GCE electrode in the ssDNA solution, and incubating for 72 hours at 4 ℃ to prepare the ssDNA/NPG/GCE electrode; wherein the sequence of the capture probe ssDNA is 5' -TGGCGGCACATTTGTCACTGCA- (CH2)3-SH-3’；
3) Preparing a BSA/ssDNA/NPG/GCE electrode of the bifunctional sensor: and (3) incubating the prepared ssDNA/NPG/GCE electrode with Bovine Serum Albumin (BSA) with the concentration of 1% m/v at 25 ℃ for 15 minutes to prepare the difunctional sensor BSA/ssDNA/NPG/GCE electrode based on the detection of the nucleic acid and the metabolic marker of the Listeria monocytogenes.
In the method for detecting listeria monocytogenes based on the bifunctional sensor for detecting nucleic acid hly and acetoin, the method for detecting nucleic acid marker hly in step (2) preferably comprises: culturing cultured Listeria monocytogenes strain 104～109CFU mL-1Taking 2mL of the solution with different concentrations, ultrasonically breaking cells and long-chain nucleic acid at the frequency of 20-25 kilohertz, and incubating the cells and the long-chain nucleic acid with a BSA/ssDNA/NPG/GCE electrode at 25 ℃ for 15 minutes to prepare an hly/BSA/ssDNA/NPG/GCE electrode; incubating hly/BSA/ssDNA/NPG/GCE electrode with 20. mu.M Methylene Blue (MB) at 25 ℃ for 5 minutes to prepare MB/hly/BSA/ssDNA/NPG/GCE electrode, and then washing 3 times with ultrapure water; putting an MB/hly/BSA/ssDNA/NPG/GCE electrode serving as a working electrode, a platinum electrode serving as a counter electrode and a saturated calomel electrode serving as a reference electrode into a reaction system of 50mM phosphate buffer solution, and performing linear sweep voltammetry by using 20mV s-1The sweep speed of the sensor is detected in a voltage range of-0.5 to + 0.1V; and (4) plotting the obtained MB peak current density value to the concentration of the Listeria monocytogenes to obtain a linear standard curve.
In the method for detecting listeria monocytogenes based on the bifunctional sensor for detecting nucleic acid hly and acetoin, the method for detecting metabolic marker acetoin in step (2) is preferably as follows: firstly, establishing a linear relation of acetoin concentration to NADH reduction amount, respectively adding 0-500 mu M acetoin into a 50mM phosphate buffer solution reaction system containing 3mM NADH according to different concentrations, taking a BSA/ssDNA/NPG/GCE electrode as a working electrode, taking a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode, and utilizing a linear sweep voltammetry method and 20mV s-1The sweep rate is detected in the voltage range of +0.1 to +0.7V, and the obtained NADH initial peak current value is used as the contrast, i.e. jNADH control(ii) a Then 1.5mg of heterologously expressed acetoin reductase is added into a buffer system to react for 10 minutes at 25 ℃, and then a BSA/ssDNA/NPG/GCE electrode is used as a working electrode and a wire is utilizedSex sweep voltammetry at 20mV s-1The sweep rate is detected within a voltage range of +0.1 to +0.7V, and the obtained peak current value of NADH after the reaction is taken as jNADH reactionWill j isNADH controlMinus jNADH reactionObtaining delta j, and drawing the delta j obtained by detecting acetoin with different concentrations to the concentration of the acetoin to obtain a linear standard curve of the concentration of the acetoin; then establishing a linear relation of acetoin concentration to Listeria monocytogenes concentration, taking 3mL of cultured Listeria monocytogenes, and culturing the Listeria monocytogenes according to the concentration of 8.0 multiplied by 105～3.5×109CFU mL-1And centrifuging at 6000rpm respectively at different concentrations, collecting culture medium supernatant, adding 50mM phosphate buffer solution to react, adding NADH with the final concentration of 3mM into the system, detecting the acetoin concentration contained in different concentrations of Listeria monocytogenes supernatant according to a method for establishing a linear relation of the acetoin concentration to the reduction amount of NADH, and drawing a linear standard curve of the acetoin concentration to the Listeria monocytogenes concentration.
The constructed BSA/ssDNA/NPG/GCE electrode is used as a dual-function sensor, and the detection of different markers (a nucleic acid marker hly and a metabolic marker acetoin) of the Listeria monocytogenes is realized by using a single sensor. The method has the outstanding advantages that the accuracy of the detection result is enhanced and the probability of false positive or false negative in the detection result obtained by detecting a single marker is reduced by detecting the markers of different layers (nucleic acid layer and metabolite layer) of the Listeria monocytogenes. The performance of the experimental test sensor shows that: the detection range of the sensor to the listeria monocytogenes is 104～109CFU mL-1(detection of nucleic acid marker) and 8.0X 105～3.5×109CFU mL-1(detection of metabolic markers).
The invention discloses a method for accurately detecting listeria monocytogenes in food by detecting different biomarkers (nucleic acid markers and metabolic markers) of the listeria monocytogenes by utilizing a bifunctional sensor, which has the prominent substantive characteristics and remarkable progress in comparison with the prior art:
1. the invention provides a method for enhancing the accuracy of detection results by detecting different layer markers of pathogenic microorganisms by using a single sensor. Traditional detection methods such as ELISA and qPCR are used for detecting single markers (protein and nucleic acid), and false positive or false negative results are easy to occur. The method for detecting different markers is integrated in a single platform, the detection of different markers of pathogenic microorganisms is realized by using one bifunctional sensor, the detection results obtained by detecting different markers of the bifunctional sensor can be mutually verified, and the accuracy of the detection results is enhanced.
2. Compared with the national standard GB4789.30-2016, the method for detecting the listeria monocytogenes does not need complex identification processes such as a kinetic experiment, a catalase experiment, sugar fermentation, a hemolysis experiment and the like, and can be used for shortening the identification time of about 2-5 days in the national standard GB4789.30-2016 to about 1.5 hours to complete the detection, so that the method has a wide application prospect.
3. The method for detecting the listeria monocytogenes has a wider detection range of 10 for the listeria monocytogenes4～109CFU mL-1(detection of nucleic acid markers, as shown in FIG. 1) and 8.0X 105～3.5×109CFU mL-1(detection of metabolic markers, as shown in FIG. 2). Meanwhile, when the dual-function sensor detects hly and acetoin, no cross reaction is caused to tested escherichia coli, pseudomonas aeruginosa, bacillus cereus, staphylococcus aureus, sphingomonas, pseudomonas putida and enterobacter sakazakii, as shown in fig. 3.
Drawings
FIG. 1 is a graph of the current density values of the bifunctional sensor for detecting the nucleic acid marker gene hly of Listeria monocytogenes with different concentrations.
Wherein: the inset is a standard curve of listeria monocytogenes concentration versus current density at the detected hly peak.
FIG. 2 is a graph of bifunctional sensors for detection of the metabolic marker acetoin.
Wherein: the graph A is a graph of acetoin concentration to NADH peak current density value, and the inset is a linear standard curve of the acetoin concentration to NADH peak current density value reduction; fig. B is a linear standard curve of acetoin concentration versus listeria monocytogenes concentration.
FIG. 3 shows the cross-reaction detection of Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, Sphingomonas, Pseudomonas putida, and Enterobacter sakazakii when hly and acetoin are detected by the dual-function sensor.
Wherein: FIG. A shows the cross-reaction detection of the above-mentioned bacteria in hly detection by the bifunctional sensor; and the graph B shows the cross reaction detection of the bifunctional sensor on the bacteria when detecting acetoin.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
In the following examples, materials, reagents, enzymes for setting the purpose of detection, and the like used are commercially available unless otherwise specified.
Example 1: preparation of bifunctional sensor based on detection of nucleic acid and metabolic marker
(1) Preparation of nanoporous gold (NPG)/Glassy Carbon Electrode (GCE): placing an Au/Ag alloy sheet with the thickness of 100nm in concentrated nitric acid, corroding for 1 hour at the temperature of 40 ℃ to prepare a nano porous gold film, fixing the prepared nano porous gold film on the surface of a glassy carbon electrode, and drying for 1-2 hours in vacuum to prepare an NPG/GCE electrode;
(2) ssDNA/NPG/GCE electrode preparation: the sequence of the capture probe (ssDNA) modified with sulfydryl at the tail end is 5' -TGGCGGCACATTTGTCACTGCA- (CH2)3-SH-3', dissolving ssDNA in ultrapure water to make a ssDNA solution with a concentration of 1 μ M, then immersing the NPG/GCE electrode in the ssDNA solution, and incubating at 4 ℃ for 72 hours to make a ssDNA/NPG/GCE electrode;
(3) preparing a BSA/ssDNA/NPG/GCE electrode of the bifunctional sensor: the ssDNA/NPG/GCE electrode was incubated with 1% (m/v) BSA at 25 ℃ for 15 minutes to produce a bifunctional sensor BSA/ssDNA/NPG/GCE electrode based on detection of Listeria monocytogenes nucleic acids and metabolic markers.
Example 2: preparation of bifunctional sensor based on detection of nucleic acid and metabolic marker
(1) Preparation of nanoporous gold (NPG)/Glassy Carbon Electrode (GCE): placing an Au/Ag alloy sheet with the thickness of 100nm in concentrated nitric acid, corroding for 2 hours at the temperature of 30 ℃ to prepare a nano porous gold film, fixing the prepared nano porous gold film on the surface of a glassy carbon electrode, and drying for 1-2 hours in vacuum to prepare an NPG/GCE electrode;
(2) ssDNA/NPG/GCE electrode preparation: the sequence of the capture probe (ssDNA) modified with sulfydryl at the tail end is 5' -TGGCGGCACATTTGTCACTGCA- (CH2)3-SH-3', dissolving ssDNA in ultrapure water to make a ssDNA solution with a concentration of 1 μ M, then immersing the NPG/GCE electrode in the ssDNA solution, and incubating at 4 ℃ for 48 hours to make a ssDNA/NPG/GCE electrode;
(3) preparing a BSA/ssDNA/NPG/GCE electrode of the bifunctional sensor: the ssDNA/NPG/GCE electrode was incubated with 2% (m/v) BSA at 4 ℃ for 30 minutes to produce a bifunctional sensor BSA/ssDNA/NPG/GCE electrode based on detection of Listeria monocytogenes nucleic acids and metabolic markers.
Example 3: preparation of bifunctional sensor based on detection of nucleic acid and metabolic marker
(1) Preparation of nanoporous gold (NPG)/Glassy Carbon Electrode (GCE): placing an Au/Ag alloy sheet with the thickness of 100nm in concentrated nitric acid, corroding for 4 hours at the temperature of 20 ℃ to prepare a nano porous gold film, fixing the prepared nano porous gold film on the surface of a glassy carbon electrode, and drying for 1-2 hours in vacuum to prepare an NPG/GCE electrode;
(2) ssDNA/NPG/GCE electrode preparation: the sequence of the capture probe (ssDNA) modified with sulfydryl at the tail end is 5' -TGGCGGCACATTTGTCACTGCA- (CH2)3-SH-3', dissolving ssDNA in ultrapure water to make a ssDNA solution with a concentration of 1 μ M, then immersing the NPG/GCE electrode in the ssDNA solution, and incubating at 4 ℃ for 60 hours to make a ssDNA/NPG/GCE electrode;
(3) preparing a BSA/ssDNA/NPG/GCE electrode of the bifunctional sensor: the ssDNA/NPG/GCE electrode was incubated with 5% (m/v) BSA at 30 ℃ for 10 minutes to produce a bifunctional sensor BSA/ssDNA/NPG/GCE electrode based on detection of Listeria monocytogenes nucleic acids and metabolic markers.
Example 4: detection of listeria monocytogenes hly gene and acetoin using bifunctional sensor based on detection of nucleic acid and metabolic marker
(1) Preparing and characterizing a bifunctional sensor standard: constructing a three-electrode detection system by using the prepared BSA/ssDNA/NPG/GCE electrode as a working electrode, a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode; wherein the BSA/ssDNA/NPG/GCE electrode was prepared as described in example 1 and previously set at 0.5M H2SO4Performing cyclic voltammetry for 20 circles, and representing the effective area of the BSA/ssDNA/NPG/GCE electrode by using reduction peak current;
(2) calibrating or calibrating the dual-function sensor: 1) detection of nucleic acid marker hly: culturing different concentrations of Listeria monocytogenes (10)4～109CFU mL-1) 2mL of each was removed, and the cells and long-chain nucleic acids were sonicated at 24 kHz and incubated with BSA/ssDNA/NPG/GCE electrodes for 15 minutes at 25 ℃ to prepare hly/BSA/ssDNA/NPG/GCE electrodes. MB/hly/BSA/ssDNA/NPG/GCE electrodes were prepared by incubating hly/BSA/ssDNA/NPG/GCE electrodes with 20. mu.M Methylene Blue (MB) at 25 ℃ for 5 minutes. Putting an MB/hly/BSA/ssDNA/NPG/GCE electrode serving as a working electrode, a platinum electrode serving as a counter electrode and a saturated calomel electrode serving as a reference electrode into a reaction system of 50mM phosphate buffer solution, and performing linear sweep voltammetry by using 20mV s-1The scanning speed of the probe is detected in a voltage range of-0.45 to 0V. And (4) plotting the obtained MB peak current density value to the concentration of the Listeria monocytogenes to obtain a linear standard curve. 2) Detection of metabolic marker acetoin: firstly, establishing a linear relation of acetoin concentration to NADH reduction amount, adding acetoin (0-500 mu M) with different concentrations into a 50mM phosphate buffer solution reaction system containing 3mM NADH, taking a BSA/ssDNA/NPG/GCE electrode as a working electrode, a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode, and utilizing a linear sweep voltammetry method to take 20mV s-1The sweep rate of (d) was measured in the range of +0.1 to +0.7V, and the obtained NADH initial peak current value was used as a control (j)NADH control). Then in a buffer systemAdding 1.5mg of heterologously expressed acetoin reductase, reacting at 25 ℃ for 10 minutes, then using a BSA/ssDNA/NPG/GCE electrode as a working electrode and using linear sweep voltammetry at 20mV s-1The sweep rate is detected within a voltage range of +0.1 to +0.7V, and the obtained peak current value of NADH after the reaction is taken as jNADH reactionWill j isNADH controlMinus jNADH reactionAnd obtaining delta j, and drawing the delta j obtained by detecting the acetoin with different concentrations to the acetoin concentration to obtain an acetoin concentration linear standard curve. Then, a linear relation of acetoin concentration to Listeria monocytogenes concentration is established, and 3mL of cultured Listeria monocytogenes (8.0 × 10) with different concentrations are taken5～3.5×109CFU mL-1) Centrifuging (6000rpm) to collect culture medium supernatant, adding 50mM phosphate buffer solution to react, simultaneously adding NADH with the final concentration of 3mM into the system, detecting the acetoin concentrations in different Listeria monocytogenes supernatants according to a method for establishing a linear relation of the acetoin concentration to the reduction amount of the NADH, and drawing a linear standard curve of the acetoin concentration to the Listeria monocytogenes concentration.
Example 5: detection of listeria monocytogenes added to milk using a dual-function sensor based on detection of nucleic acids and metabolic markers
(1) Preparing and characterizing a bifunctional sensor standard: constructing a three-electrode detection system by using the prepared BSA/ssDNA/NPG/GCE electrode as a working electrode, a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode; wherein the BSA/ssDNA/NPG/GCE electrode was prepared as described in example 3 and previously set at 0.5M H2SO4Performing cyclic voltammetry for 20 circles, and representing the effective area of the BSA/ssDNA/NPG/GCE electrode by using reduction peak current;
(2) calibrating or calibrating the dual-function sensor: drawing a corresponding linear standard curve according to the detection of the nucleic acid marker hly and the detection of acetoin in example 4, wherein the corresponding detection results are shown in FIG. 1 and FIG. 2;
(3) detecting the concentration of listeria monocytogenes in milk by using a standard-adding recovery method: 25mL of finished milk was added to 225mL of media suitable for growth of Listeria monocytogenes (designated as solution 1). 0.1mL of the solution was taken out of the solution 1 and added10mL of a medium suitable for growth of Listeria monocytogenes (designated solution 2). Adding appropriate amount of Listeria monocytogenes to give final concentration of 104CFU mL-1Culturing at 37 ℃ for 6 hours, 9 hours and 12 hours, detecting the nucleic acid marker and the metabolic marker of the listeria monocytogenes by using the BSA/ssDNA/NPG/GCE electrode prepared in the step (1) according to the method in the step (2), and respectively bringing the obtained peak current values into different linear standard curves to correspondingly obtain the concentration of the listeria monocytogenes, wherein the obtained concentration of the listeria monocytogenes has no larger deviation compared with a plate counting method, and the results are shown in Table 1.
TABLE 1
a: detecting the concentration of listeria monocytogenes obtained by hly; b: the listeria monocytogenes concentration obtained by detecting acetoin.
Claims (4)
1. A method for detecting Listeria monocytogenes based on a bifunctional sensor for detecting nucleic acid hly and acetoin comprises the following steps:
(1) preparing a bifunctional sensor based on detection of listeria monocytogenes nucleic acid and metabolic markers;
(2) calibrating or calibrating the dual-function sensor;
(3) taking a milk sample as an example, detecting the concentration of listeria monocytogenes in milk by using a standard-adding recovery method based on a dual-function sensor;
the method is characterized in that:
the preparation method of the bifunctional sensor based on detection of the listeria monocytogenes nucleic acid and the metabolic marker in the step (1) comprises the following steps:
1) preparing a nanoporous gold (NPG)/Glassy Carbon Electrode (GCE): placing an Au/Ag alloy sheet with the thickness of 100 +/-10 nm in concentrated nitric acid, corroding for 1 hour at the temperature of 40 +/-2 ℃ to prepare a nano porous gold film, fixing the prepared nano porous gold film on the surface of a glassy carbon electrode, and drying for 1-2 hours in vacuum to prepare an NPG/GCE electrode;
2) preparation of ssDNA/NPG/GCE electrodes: dissolving ssDNA (single-stranded deoxyribonucleic acid) of a capture probe which is partially complementary with an hly gene and is modified with sulfydryl at the tail end into ultrapure water to prepare a ssDNA solution with the concentration of 0.5-3 mu M, then immersing the NPG/GCE electrode into the ssDNA solution, and incubating for 48-72 hours at 4 ℃ to prepare the ssDNA/NPG/GCE electrode; wherein the sequence of the capture probe ssDNA is 5' -TGGCGGCACATTTGTCACTGCA- (CH2)3-SH-3’；
3) Preparing a BSA/ssDNA/NPG/GCE electrode of the bifunctional sensor: incubating the prepared ssDNA/NPG/GCE electrode and Bovine Serum Albumin (BSA) with the concentration of 0.5-5% m/v at 4-30 ℃ for 15-30 minutes to prepare a difunctional sensor BSA/ssDNA/NPG/GCE electrode based on detection of Listeria monocytogenes nucleic acid and metabolic markers;
the method for calibrating or calibrating the dual-function sensor in the step (2) comprises the following steps:
1) detection of nucleic acid marker hly: the cultured concentration is 104～109CFU mL-1Taking 1-10 mL of the listeria monocytogenes liquid according to different concentrations, ultrasonically breaking cells and long-chain nucleic acid at the frequency of 20-60 kilohertz, and incubating the cells and the long-chain nucleic acid with a BSA/ssDNA/NPG/GCE electrode for 5-30 minutes at the temperature of 20-30 ℃ to prepare a hly/BSA/ssDNA/NPG/GCE electrode; incubating the hly/BSA/ssDNA/NPG/GCE electrode and 5-40 mu M methylene blue for short MB at 20-30 ℃ for 5-30 minutes to prepare an MB/hly/BSA/ssDNA/NPG/GCE electrode; putting an MB/hly/BSA/ssDNA/NPG/GCE electrode serving as a working electrode, a platinum electrode serving as a counter electrode and a saturated calomel electrode serving as a reference electrode into a reaction system of 50mM phosphate buffer solution, and performing linear sweep voltammetry by using 20-100 mV s-1Detecting the scanning speed in a voltage range of-0.5 to +0.1V, and drawing the obtained MB peak current density value to the concentration of the Listeria monocytogenes to obtain a linear standard curve;
2) detection of metabolic marker acetoin: firstly, establishing a linear relation of acetoin concentration to NADH reduction amount, adding the acetoin into a 50mM phosphate buffer solution reaction system containing 1-5 mM NADH according to different concentrations of 0-500 mu M, taking a BSA/ssDNA/NPG/GCE electrode as a working electrode, a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode, and utilizing a linear sweep voltammetry method to obtain a sample with the concentration of 20-100 mV s-1The sweep rate of (1) is detected in the voltage range of +0.1 to +0.7V, and the obtained NADH initial peak current value is used as a reference, i.e. jNADH control(ii) a Then adding 1-5 mg of heterologously expressed acetoin reductase into a buffer system, reacting for 5-30 minutes at 20-30 ℃, then taking a BSA/ssDNA/NPG/GCE electrode as a working electrode, and utilizing a linear scanning voltammetry to obtain a sample with the concentration of 20-100 mV s-1The sweep rate is detected within a voltage range of +0.1 to +0.7V, and the obtained peak current value of NADH after the reaction is taken as jNADH reactionWill j isNADH controlMinus jNADH reactionObtaining delta j, and drawing the delta j obtained by detecting acetoin with different concentrations to the concentration of the acetoin to obtain a linear standard curve of the concentration of the acetoin; then establishing a linear relation of acetoin concentration to Listeria monocytogenes concentration, and taking 1-50 mL of cultured solution with the concentration of 8.0 multiplied by 105～3.5×109CFU mL-1Centrifuging bacterium solutions with different concentrations at 4000-8000 rpm, collecting culture medium supernatant, adding a 50mM phosphate buffer solution reaction system, adding NADH with the final concentration of 1-5 mM into the system, detecting the acetoin concentration in the listeria monocytogenes supernatant with different concentrations according to a method for establishing a linear relation of the acetoin concentration to the NADH reduction amount, and drawing a linear standard curve of the acetoin concentration to the listeria monocytogenes concentration;
taking the milk sample as an example in the step (3), the method for detecting the concentration of listeria monocytogenes in milk by using a standard-adding recovery method based on the dual-function sensor comprises the following steps:
adding 25mL of finished milk into 225mL of culture medium suitable for growth of Listeria monocytogenes, and naming the solution as solution 1; 0.1mL of the solution was removed from solution 1 and added to 10mL of a medium suitable for growth of Listeria monocytogenes, designated solution 2; adding appropriate amount of Listeria monocytogenes into the solution 2 to make the final concentration of the bacteria be 104CFU mL-1Culturing at 37 ℃ for 6 hours, 9 hours and 12 hours, detecting the nucleic acid marker and the metabolic marker of the listeria monocytogenes by using the BSA/ssDNA/NPG/GCE electrode prepared in the step (1) according to the method in the step (2), and respectively substituting the obtained peak current values into different linear standard curves to correspondingly obtain the concentration of the listeria monocytogenes.
2. The method for detecting listeria monocytogenes based on the bifunctional sensor for detecting nucleic acids hly and acetoin according to claim 1, wherein the method for preparing the bifunctional sensor for detecting listeria monocytogenes nucleic acids and metabolic markers in step (1) is as follows:
1) preparing a nanoporous gold (NPG)/Glassy Carbon Electrode (GCE): placing an Au/Ag alloy sheet with the thickness of 100nm in concentrated nitric acid, corroding for 1 hour at the temperature of 40 ℃ to prepare a nano porous gold film, fixing the prepared nano porous gold film on the surface of a glassy carbon electrode, and drying for 1 hour in vacuum to prepare an NPG/GCE electrode;
2) preparation of ssDNA/NPG/GCE electrodes: dissolving ssDNA (single stranded deoxyribonucleic acid) of a capture probe which is partially complementary with an hly gene and is modified with sulfydryl at the tail end in ultrapure water to prepare a ssDNA solution with the concentration of 1 mu M, then immersing an NPG/GCE electrode in the ssDNA solution, and incubating for 72 hours at 4 ℃ to prepare the ssDNA/NPG/GCE electrode; wherein the sequence of the capture probe ssDNA is 5' -TGGCGGCACATTTGTCACTGCA- (CH2)3-SH-3’；
3) Preparing a BSA/ssDNA/NPG/GCE electrode of the bifunctional sensor: and (3) incubating the prepared ssDNA/NPG/GCE electrode with Bovine Serum Albumin (BSA) with the concentration of 1% m/v at 25 ℃ for 15 minutes to prepare the difunctional sensor BSA/ssDNA/NPG/GCE electrode based on the detection of the nucleic acid and the metabolic marker of the Listeria monocytogenes.
3. The method for detecting listeria monocytogenes based on the bifunctional sensor for detecting nucleic acids hly and acetoin according to claim 1, wherein the method for detecting the nucleic acid marker hly in step (2) is: culturing cultured Listeria monocytogenes strain 104～109CFU mL-1Taking 2mL of the solution with different concentrations, ultrasonically breaking cells and long-chain nucleic acid at the frequency of 20-25 kilohertz, and incubating the cells and the long-chain nucleic acid with a BSA/ssDNA/NPG/GCE electrode at 25 ℃ for 15 minutes to prepare an hly/BSA/ssDNA/NPG/GCE electrode; incubating hly/BSA/ssDNA/NPG/GCE electrode with 20 mu M methylene blue abbreviated as MB at 25 ℃ for 5 minutes to prepare MB/hly/BSA/ssDNA/NPG/GCE electrode, and then washing with ultrapure water for 3 times; MB/hly/BSA/ssDNA/NPG/GCE electrode is used as a working electrode, so as toThe platinum electrode is used as a counter electrode, the saturated calomel electrode is used as a reference electrode, the counter electrode is put into a reaction system of 50mM phosphate buffer solution, and the linear sweep voltammetry is adopted with the s of 20mV-1The sweep speed of the sensor is detected in a voltage range of-0.5 to + 0.1V; and (4) plotting the obtained MB peak current density value to the concentration of the Listeria monocytogenes to obtain a linear standard curve.
4. The method for detecting listeria monocytogenes based on the bifunctional sensor for detecting nucleic acids hly and acetoin according to claim 1, wherein the metabolic marker acetoin in step (2) is detected by: firstly, establishing a linear relation of acetoin concentration to NADH reduction amount, respectively adding 0-500 mu M acetoin into a 50mM phosphate buffer solution reaction system containing 3mM NADH according to different concentrations, taking a BSA/ssDNA/NPG/GCE electrode as a working electrode, taking a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode, and utilizing a linear sweep voltammetry method and 20mV s-1The sweep rate of (1) is detected in the voltage range of +0.1 to +0.7V, and the obtained NADH initial peak current value is used as a reference, i.e. jNADH control(ii) a Then 1.5mg of heterologously expressed acetoin reductase is added into a buffer system to react for 10 minutes at 25 ℃, and then a BSA/ssDNA/NPG/GCE electrode is used as a working electrode and linear sweep voltammetry is used to obtain a solution with the concentration of 20mV s-1The sweep rate is detected within a voltage range of +0.1 to +0.7V, and the obtained peak current value of NADH after the reaction is taken as jNADH reactionWill j isNADH controlMinus jNADH reactionObtaining delta j, and drawing the delta j obtained by detecting acetoin with different concentrations to the concentration of the acetoin to obtain a linear standard curve of the concentration of the acetoin; then establishing a linear relation of acetoin concentration to Listeria monocytogenes concentration, taking 3mL of cultured Listeria monocytogenes, and culturing the Listeria monocytogenes according to the concentration of 8.0 multiplied by 105～3.5×109CFU mL-1Centrifuging at 6000rpm respectively at different concentrations, collecting culture medium supernatant, adding 50mM phosphate buffer solution to react, adding NADH with final concentration of 3mM into the system, detecting acetoin concentration in different concentrations of Listeria monocytogenes supernatant according to a method for establishing a linear relation of the acetoin concentration to NADH reduction amount, and drawing a linear standard curve of the acetoin concentration to the Listeria monocytogenes concentrationA wire.
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