CN114791491A - Method for rapidly detecting staphylococcus aureus by using biomembrane interference technology - Google Patents

Abstract

The invention discloses a method for rapidly detecting staphylococcus aureus by utilizing a biomembrane interference technology, which comprises the following steps: step one, sensor balancing; secondly, activating a sensor; step three, immobilizing the antibody; step four, sealing once; step five, secondary sealing; and step six, sample detection and result analysis. The invention has the beneficial effects that: the method has the advantages of few steps, no mark, real-time monitoring, simplicity, convenience and rapidness, and simple result judgment; the operation is simple and easy to learn, the prepared reagent and the prepared sample are only added into the sample plate to be detected on a computer, and the existence of staphylococcus aureus can be judged by reading the binding signal in real time; the antibody diluted by the acetic acid-sodium acetate buffer solution can be recycled, so that the detection cost is reduced; the high flux is detected, and the simultaneous detection of up to 95 samples can be realized by matching with a corresponding biomolecule interaction instrument and a matched sample plate; has better specificity, and can eliminate the interference of non-target bacteria in the sample.

Description

Method for rapidly detecting staphylococcus aureus by using biomembrane interference technology

Technical Field

The invention relates to a rapid detection method of staphylococcus aureus, in particular to a rapid detection method of staphylococcus aureus by combining an antibody with a biomembrane interference technology.

Background

Staphylococcus aureus is a facultative anaerobic gram-positive bacterium widely present in nature and on human skin, and is a common pathogenic bacterium of skin suppurative infection, and enterotoxin produced by staphylococcus aureus can cause poisoning. The food is easy to be polluted, which causes food poisoning of human body, and the main symptoms are fever, diarrhea, nausea and vomiting. Staphylococcus aureus secretes more than 20 kinds of toxic proteins, the common types are 7, the staphylococcus aureus can resist high temperature, and the staphylococcus aureus can not be damaged when being heated for 30min at 100 ℃. It has been reported that adult consumption of only about 100ng of enterotoxin a can cause food poisoning. In recent years, food poisoning caused by staphylococcus aureus has been the 4 th worldwide. At present, the traditional staphylococcus aureus detection method has the advantages of high sensitivity and low cost, but the steps of selective pre-enrichment, selective plate culture, staining microscopy, plasma coagulase identification and the like are complex and long in time consumption, and the requirement for rapid detection cannot be met. The immunological detection method (such as enzyme-linked immunosorbent assay) has obviously shortened detection time, but has high selectivity on reagents. Therefore, in order to reduce the occurrence of related food-borne diseases, a novel detection method for staphylococcus aureus, which is rapid, convenient, sensitive, accurate and easy to operate, is urgently needed to be developed.

The biofilm interference (BLI) technique is a technique for analyzing or detecting interaction between biomolecules by monitoring changes in optical interference signals in real time. The Octet used by the technology is a multi-channel detection device, the whole detection process is fully automatic, the temperature of a detection module can be controlled, and a detected sample can be recovered. The technology has the advantages of high sensitivity, no microfluidic system, no mark, rapidness, simplicity, convenience, capability of providing a detection result in real time and the like, and is very suitable for rapidly and sensitively detecting the food-borne pathogenic bacteria.

The antibody is an identification element with the widest application range in the biosensor, and can be divided into a polyclonal antibody, a monoclonal antibody and a genetic engineering antibody according to the preparation method and the principle of the antibody. The use of antibodies for antigen recognition is known as an immunoassay, which is used in a variety of biosensor formats. However, it is only reported that the antibody is used as a biological recognition element of the BLI biosensor and the technology is applied to the rapid detection of the food-borne pathogenic bacteria.

The invention takes the antibody as the biological recognition element of the second generation amino coupling sensor to carry out recognition detection on the staphylococcus aureus. The second generation of amino coupled sensors has increased binding density, more stable curing conditions, and greatly reduced non-specific binding. The sensor surface is adaptable to a variety of pH and salt conditions, providing robustness and flexibility for regeneration condition development for higher throughput applications. The surface of the second generation amino coupling sensor is modified with carboxyl groups, and when an antibody is fixed, the carboxyl groups on the surface of the sensor are activated in an activating reagent mixed by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form NHS ester; the NHS ester can react with the amino group on the antibody to form an extremely stable amide bond to immobilize the antibody on the second generation amino-coupled sensor surface. If staphylococcus aureus exists in the sample, the thalli can be specifically identified and combined by the antibody fixed on the surface, so that the thickness of the optical layer of the sensor is increased, the interference spectrum generates displacement, and the generated response signal is detected and recorded by the spectrometer in real time, thereby realizing the real-time label-free rapid detection of the staphylococcus aureus.

Disclosure of Invention

The invention aims to provide a rapid detection method for staphylococcus aureus based on a biomembrane interference technology, which aims to solve the problems of multiple steps, complex operation and the like in the traditional detection method for staphylococcus aureus.

The invention provides a method for quickly detecting staphylococcus aureus by combining an antibody with a biofilm interference technology. The method comprises sensor balance, sensor activation, antibody immobilization, primary sealing, secondary sealing, sample detection and result analysis, and comprises the following specific steps:

step one, sensor balancing: firstly, immersing the tail end of a second generation amino coupling sensor into pure water for prewetting for at least 10 minutes to balance the sensor;

step two, sensor activation: immersing the second generation amino coupled sensor after the balance into a mixed reagent of 20mM 1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride and 10mM N-hydroxysuccinimide for activation for 300-450 seconds;

step three, antibody immobilization: immersing the activated second generation amino coupled sensor into an antibody solution with the concentration of 12.5-100 mu g/mL and diluted by acetic acid-sodium acetate buffer solution with the pH value of 5-7 for immobilization for 400-900 seconds;

step four, closing the ethanolamine hydrochloride solution for one time: immersing the second generation amino coupled sensor fixed with the antibody into ethanolamine hydrochloride solution (pH 8.5, 1mol/L) for blocking for 300-450 seconds;

step five, secondary sealing of bovine serum albumin solution: immersing the second generation amino coupling sensor after ethanolamine hydrochloride blocking into 1% bovine serum albumin solution for blocking for 300-450 seconds;

step six, sample detection and result analysis: after blocking in bovine serum albumin solution, immersing the second generation amino coupled sensor in PBS phosphate buffer solution containing 0.02% Tween-20 for balancing for 150-450 seconds, then immersing the second generation amino coupled sensor in a sample for detecting staphylococcus aureus, reading a binding signal in real time, and analyzing a detection result according to the binding signal.

The invention has the beneficial effects that:

the rapid detection method for staphylococcus aureus based on the biomembrane interference technology provided by the invention has the advantages of few steps, no mark, real-time monitoring, simplicity, convenience and rapidness, and simple result judgment; the operation is simple and easy to learn, the prepared reagent and the prepared sample are only added into the sample plate to be detected on the computer, and the existence of the staphylococcus aureus can be judged by reading the binding signal in real time; the antibody diluted by the acetic acid-sodium acetate buffer solution can be recycled, so that the detection cost is reduced; the high flux is detected, and the simultaneous detection of up to 95 samples can be realized by matching with a corresponding biomolecule interaction instrument and a matched sample plate; has better specificity, and can eliminate the interference of non-target bacteria in the sample.

Drawings

FIG. 1 is a schematic diagram of the specific detection result of the detection method of the present invention.

FIG. 2 is a schematic diagram of the detection of Staphylococcus aureus of different concentrations in the detection method of the present invention.

Detailed Description

Specific examples are written below and more detailed implementation procedures are given, all examples are carried out according to the present technical scheme, and experimental methods without specific conditions noted in the examples are generally carried out according to conventional conditions or according to conditions recommended by reagent manufacturers.

The main materials and reagents used in the examples are shown in Table 1, and the main equipment is shown in Table 2.

TABLE 1 Main materials and reagents information Table

TABLE 2 Main Instrument information Table

Example 1: investigation of the specificity of the present protocol

The method is characterized by comprising the following steps of (1) carrying out detection analysis by taking staphylococcus aureus as a target bacterium and salmonella, escherichia coli, bacillus subtilis, pseudomonas aeruginosa and listeria monocytogenes as non-target bacteria:

(1) inoculating Staphylococcus aureus, Salmonella, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Listeria monocytogenes to the strainSterilized tryptone soy broth was cultured at 37 ℃ for 15 to 18 hours with shaking at 200 rpm. Centrifuging the bacterial suspension, discarding supernatant, washing twice with PBS phosphate buffer solution, and adjusting the bacterial suspension concentration of six bacteria to 1 × 10 with PBS phosphate buffer solution containing 0.02% Tween-20 ⁶ CFU/mL, to be tested.

(2) Pure water, a mixed reagent of 20mM 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 10mM N-hydroxysuccinimide, an antibody solution with a concentration of 25 mu g/mL diluted by an acetic acid-sodium acetate buffer solution with a pH of 6, an ethanolamine hydrochloride solution (pH 8.5, 1mol/L), a 1% bovine serum albumin solution, a PBS phosphate buffer solution containing 0.02% Tween-20 and a sample to be detected are sequentially added to corresponding positions of a 96-well plate.

(3) The second generation amino-coupled sensors were hydrated (sensor ends immersed in pure water) for 9 minutes in advance, and the loaded 96-well plate was then placed in an Octet Red 96 molecular interaction instrument and the system was run. The second generation of amino-coupled sensors were sequentially immersed in pure water (60 seconds), a mixed reagent of 20mM 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 10mM N-hydroxysuccinimide (300 seconds), an antibody solution diluted with a pH 6 acetic acid-sodium acetate buffer solution at a concentration of 25. mu.g/mL (400 seconds), an ethanolamine hydrochloride solution (300 seconds), a 1% bovine serum albumin solution (300 seconds), a PBS phosphate buffer solution containing 0.02% Tween-20 (180 seconds), and a sample to be measured (900 seconds).

(4) The instrument can observe and detect the signal value in real time when in operation, as shown in figure 1, the signal value of a target bacterium staphylococcus aureus sample is far above the effective signal value (0.0075nm), and the signal values of non-target bacteria salmonella, escherichia coli, bacillus subtilis, pseudomonas aeruginosa and listeria monocytogenes are all negative values, which indicates that the method has higher specificity.

Example 2: detection of different concentrations of Staphylococcus aureus

(1) Staphylococcus aureus was inoculated to sterilized tryptone soy broth with an inoculating loop and cultured at 37 ℃ for 15-18 hours with shaking at 200 rpm. Centrifuging the bacterial suspension, discarding the supernatant, and washing with PBS phosphate buffer solutionNext, the concentration of the bacterial suspensions was adjusted to 10 using PBS phosphate buffer solution containing 0.02% Tween-20 ⁷ CFU/mL、10 ⁶ CFU/mL、10 ⁵ CFU/mL, to be tested.

(2) The procedure of (3) was the same as those of (2) and (3) in example 1.

(4) The detection signal value can be observed in real time when the instrument is operated, as shown in fig. 2, the detection signal is obviously and positively correlated with the concentration of the staphylococcus aureus, which indicates that the method not only can be used for qualitative detection of the staphylococcus aureus, but also can be used for quantitative detection of the staphylococcus aureus.

Claims (1)

1. A method for rapidly detecting staphylococcus aureus by utilizing a biomembrane interference technology is characterized by comprising the following steps: the method comprises the following steps of sensor balance, sensor activation, antibody immobilization, primary sealing, secondary sealing, sample detection and result analysis, and specifically comprises the following steps:

step one, sensor balancing: pre-wetting the second generation amino-coupled sensor ends in pure water for at least 10 minutes to equilibrate the sensors;

step two, sensor activation: immersing the second generation amino coupled sensor after the balance into a mixed reagent of 20mM 1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride and 10mM N-hydroxysuccinimide for activation for 300-450 seconds;

step three, antibody immobilization: immersing the activated second generation amino coupled sensor into an antibody solution with the concentration of 12.5-100 mu g/mL and diluted by acetic acid-sodium acetate buffer solution with the pH value of 5-7 for immobilization for 400-900 seconds;

step four, closing the ethanolamine hydrochloride solution for one time: immersing the second generation amino coupled sensor fixed with the antibody into ethanolamine hydrochloride solution (pH 8.5, 1mol/L) for blocking for 300-450 seconds;

step five, secondary sealing of the bovine serum albumin solution: immersing the second generation amino coupling sensor after ethanolamine hydrochloride blocking into 1% bovine serum albumin solution for blocking for 300-450 seconds;

step six, sample detection and result analysis: after blocking in bovine serum albumin solution, immersing the second generation amino coupled sensor in PBS phosphate buffer solution containing 0.02% Tween-20 for balancing for 150-450 seconds, then immersing the second generation amino coupled sensor in a sample for detecting staphylococcus aureus, reading a binding signal in real time, and analyzing a detection result according to the binding signal.

Images